Publications (155)

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    [Show abstract] [Hide abstract] ABSTRACT: Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS), however none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ-line precursors. The structures of antigen binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the CDR H2 loops that binds terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ-line origin), that are nevertheless complementary both in shape and charge to the antigen. S55- 3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains while the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies.
    Full-text available · Article · Mar 2016 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract] ABSTRACT: Septic shock is a leading cause of death, and, 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 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 unliganded and in complex with lipid A carbohydrate backbone. 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, while 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 towards 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 ssDNA fragments, which may provide clues into the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Article · Jun 2015 · Journal of Biological Chemistry
  • Conference Paper · Nov 2014
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    File available · Data · Apr 2014
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    [Show abstract] [Hide abstract] ABSTRACT: The structure of the antigen-binding fragment of mAb S25-26 determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)-Kdo(2→4)Kdo (Kdo=3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) displays a germline-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs, despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germline residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three dimensional structure of an αGal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as they have been implicated in allergic response in patients receiving therapeutic antibodies.
    Full-text available · Article · Mar 2014 · Journal of Biological Chemistry
  • Walter Richter · Lena Heinbockel · Yani Kaconis · [...] · Klaus Brandenburg
    [Show abstract] [Hide abstract] ABSTRACT: We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope-in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex.
    Article · Feb 2013 · Innate Immunity
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    [Show abstract] [Hide abstract] ABSTRACT: The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2→4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2. The structure reveals that the modification of the Kdo residue at position 5 results in a rearrangement of intramolecular hydrogen bonds in the antigen that allows it to assume a novel conformation in the antibody-combining site. The cross-reactive binding of S25-2 to this synthetic ligand highlights the adaptability of this antibody to non-natural synthetic analogues.
    Full-text available · Article · Jan 2013 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
  • [Show abstract] [Hide abstract] ABSTRACT: Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.
    Article · Nov 2012 · Proceedings of the National Academy of Sciences
  • Lore Brade · Selma Hensen · Helmut Brade
    [Show abstract] [Hide abstract] ABSTRACT: The immune response of cows against the core oligosaccharide of Escherichia coli rough mutants (core types R1-R4, K-12 and J-5) was investigated after immunization with a synthetic glycoconjugate composed of deacylated LPS conjugated to hemocyanine (22 animals). Ab formation was measured by ELISA using LPS or deacylated LPS conjugated to BSA as an Ag. The glycoconjugate immunogens were used to vaccinate cows (36 animals), which were then challenged intramammarily with E. coli O 157 (K1 negative, R1 core type). Compared with control groups no protection was observed, although high titers against the R1 core type were detected in vaccinated animals. Western blots using the immune sera showed that the Ab response was directed against the core region and not against the O-antigen, which may explain the failure of the vaccine.
    Article · Oct 2012 · Innate Immunity
  • Conference Paper · Nov 2011
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    [Show abstract] [Hide abstract] ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
    Full-text available · Conference Paper · Nov 2011
  • Conference Paper · Nov 2011
  • Lore Brade · Holger Heine · Satish Raina · [...] · Sven Müller-Loennies
    [Show abstract] [Hide abstract] ABSTRACT: The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200 d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.
    Article · Aug 2011 · Innate Immunity
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    [Show abstract] [Hide abstract] ABSTRACT: The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.
    Full-text available · Article · May 2011 · Glycobiology
  • [Show abstract] [Hide abstract] ABSTRACT: The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å. The antigen-binding site of this antibody differs from the well-characterized antibody S25-2 by a single mutation away from the germline of asparagine H53 to lysine, yet this one mutation results in a significant increase in avidity across a range of antigens. A comparison of the two antibody structures reveals that the mutated Lys H53 forms additional hydrogen bonds and/or charged-residue interactions with the second Kdo residue of every antigen having two or more carbohydrate residues. Significantly, the NH53K mutation results from a single nucleotide substitution in the germline sequence common among a panel of antibodies raised against glycoconjugates containing carbohydrate epitopes of chlamydial LPS. Like S25-2, S25-39 displays significant induced fit of complementarity determining region (CDR) H3 upon antigen binding, with the unliganded structure possessing a conformation distinct from those reported earlier for S25-2. The four different observed conformations for CDR H3 suggest that this CDR has evolved to exploit the recognition potential of a flexible loop while minimizing the associated entropic penalties of binding by adopting a limited number of ordered conformations in the unliganded state. These observations reveal strategies evolved to balance adaptability and specificity in the germline antibody response to carbohydrate antigens.
    Article · Mar 2011 · Biochemistry
  • [Show abstract] [Hide abstract] ABSTRACT: Synthesis of the title trisaccharide was accomplished by sugar chain extension starting from the non-reducing terminus: coupling of GlcpNAc with ld-Hepp, then adding Glcp-OAll. An alternative route started from the reducing end: coupling of ld-Hepp with Glcp-OAll, then addition of GlcpNAc. In the synthesis of the title disaccharide a modification of the first approach was employed. The allyl glycosides were coupled with cysteamine, activated with thiophosgene and conjugated to bovine serum albumin (BSA). The neoglycoconjugates obtained were used in immunochemical studies of monoclonal and polyclonal antibodies directed against Escherichia coli K-12 lipopolysaccharide.
    Article · Jul 2010 · ChemInform
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    [Show abstract] [Hide abstract] ABSTRACT: Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a KD of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.
    Full-text available · Article · Dec 2009 · Glycobiology
  • Kai Schmengler · Torsten Goldmann · Lore Brade · [...] · Ulrich Zähringer
    [Show abstract] [Hide abstract] ABSTRACT: The lipopolysaccharide (LPS) of Pseudomonas aeruginosa has been identified to contain an inner-core structure expressing a Pseudomonas-specific epitope. This target structure is characterized by a highly phosphorylated and 7-O-carbamoyl-l-glycero-alpha-d-manno-heptopyranose (CmHep) and was found to be present in all human-pathogenic Pseudomonas species of the Palleroni (RNA)-classification I scheme. We raised and selected the monoclonal antibody S60-4-14 (mAb S60-4-14, subtype IgG1) from mice immunized with heat-killed Pseudomonas bacteria. The epitope of this mAb was found to reside in the inner-core structure of P. aeruginosa and, hence, successfully evaluated for the immunohistochemical detection of P. aeruginosa in formalin- or HOPE-fixed (Hepes-glutamic acid buffer-mediated organic solvent protection effect) and paraffin-embedded human lung tissue slices. Lung specimens, mainly from explanted lungs of cystic fibrosis (CF) patients, as well as P. aeruginosa isolates from patients suffering from CF and patients with extrapulmonar Pseudomonas infections were investigated by PCR, immunohistochemistry, and Western blot analysis with mAb S60-4-14. The results revealed an unequivocal coincidence of PCR and immunohistochemistry. Together with the Western blot results mAb S60-4-14 displays a potential diagnostic tool for the specific identification of P. aeruginosa in infected lungs of CF.
    Article · Dec 2009 · European journal of cell biology
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    [Show abstract] [Hide abstract] ABSTRACT: The structures of antigen-binding fragments from two related monoclonal antibodies have been determined to high resolution in the presence of several carbohydrate antigens raised against chlamydial lipopolysaccharide. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes and how unrelated CDR H3 sequences can result in convergent binding of clinically relevant bacterial antigens.
    Full-text available · Article · Dec 2009 · Biochemistry
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    [Show abstract] [Hide abstract] ABSTRACT: In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, the comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.
    Full-text available · Article · Sep 2009 · Glycobiology