Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26.

The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

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.

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.

Groove-type recognition of chlamydiaceae-specific lipopolysaccharide antigen by a family of antibodies possessing an unusual variable heavy chain N-linked glycan.

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 germ-line-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 germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity.

Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: incorporation of glucuronic acid and phosphoethanolamine in the heptose region.

It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose.

Exploring the cross-reactivity of S25-2: complex with a 5,6-dehydro-Kdo disaccharide.

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.

Intact rough- and smooth-form lipopolysaccharides from Escherichia coli separated by preparative gel electrophoresis exhibit differential biologic activity in human macrophages.

We established a new preparative separation procedure, based on DOC/PAGE, to isolate intact lipopolysaccharide (LPS) fractions from natural LPS preparations of Escherichia coli. Analysis of the chemical integrity of LPS fractions by MS showed that no significant chemical modifications were introduced by the procedure. Contamination with toll-like receptor 2 (TLR2)-reactive cell-wall components present in the natural LPS mixture was effectively removed by the procedure, as determined by the absence of reactivity of the purified fractions in a HEK293-TLR2 cell line.

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS.

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.

Evaluation of a LPS-based glycoconjugate vaccine against bovine Escherichia coli mastitis: Formation of LPS Abs in cows after immunization with E. coli core oligosaccharides conjugated to hemocyanine.

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.

Molecular basis of lipopolysaccharide heterogeneity in Escherichia coli: envelope stress-responsive regulators control the incorporation of glycoforms with a third 3-deoxy-α-D-manno-oct-2-ulosonic acid and rhamnose.

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes.

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits.

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.

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia.

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.

A common NH53K mutation in the combining site of antibodies raised against chlamydial LPS glycoconjugates significantly increases avidity.

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 lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type.

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection.

Efficient Synthesis of 4-Amino-4-deoxy-L-arabinose and Spacer-equipped 4-Amino-4-deoxy-L-arabinopyranosides by Transglycosylation Reactions.

Methyl 4-azido-4-deoxy-β-L-arabinopyranoside has been synthesized in five steps starting from methyl β-D-xylopyranoside in a multigram scale without chromatographic purification in 78% overall yield. The transformation relied on selective tosylation/nosylation at O-4 followed by acylation, S(N)2 displacement with sodium azide and subsequent deprotection. The methyl 4-azido-4-deoxy-arabinoside was then converted into allyl, propenyl, ω-bromohexyl and chlorethoxyethyl spacer glycosides by transglycosylation with the respective alcohols in good yields and fair anomeric selectivity.

Synthesis of a neoglycoconjugate containing a Chlamydophila psittaci-specific branched Kdo trisaccharide epitope.

The branched Kdo trisaccharide sodium (3-deoxy-alpha-D-manno-oct-2-ulopyranosyl)onate-(2-->8)-[sodium (3-deoxy-alpha-D-manno-oct-2-ulopyranosyl)onate-(2-->4)]-sodium (allyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate has been prepared utilizing the regioselective glycosylation of the C-7, C-8 diol entity of a Kdo monosaccharide acceptor with a Kdo bromide donor followed by the attachment of the third Kdo unit to O-4 of the disaccharide intermediate. Deacetylation and hydrolysis of the methyl ester groups furnished the trisaccharide allyl glycoside which was converted into the corresponding 3-(2-aminoethylthio)propyl glycoside. Subsequent covalent attachment to bovine serum albumin furnished a neoglycoconjugate serving as an antigen for the induction of Chlamydophila psittaci-specific monoclonal antibodies.

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci.

Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.

Monoclonal antibody S60-4-14 reveals diagnostic potential in the identification of Pseudomonas aeruginosa in lung tissues of cystic fibrosis patients.

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.

Antibodies raised against chlamydial lipopolysaccharide antigens reveal convergence in germline gene usage and differential epitope recognition.

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.

Glycosyltransferases involved in the biosynthesis of the inner core region of different lipopolysaccharides.

The inner core of lipopolysaccharide (LPS) structures in Gram-negative bacteria is considered a highly conserved region. The sugar connecting the membrane-associated lipid A moiety with the hydrophilic saccharide moiety, 3-deoxy-alpha-d-manno-oct-2-ulosonic acid (Kdo) is present in every LPS molecule investigated but it may be partially replaced by d-glycero-alpha-d-talo-oct-2-ulosonic acid (Ko). l-Glycero-alpha-d-manno-heptose (Hep) and phosphate residues are part of most but not all LPS structures and additionally, modifications with 4-amino-4-deoxy-beta-l-arabinose (Ara4N) residues occur in some.

Critical role for interleukin-1beta (IL-1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL-1beta in mouse macrophages.

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection.

The role of CDR H3 in antibody recognition of a synthetic analog of a lipopolysaccharide antigen.

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.

Synthesis and antigenic properties of C-7-modified Kdo mono- and disaccharide ligands and Kdo disaccharide interresidue lactones.

In order to define binding interactions of Kdo-specific monoclonal antibodies directed against the chlamydial alpha-(2-->8)-linked Kdo disaccharide epitope on a molecular level, modifications at the 7-position of the proximal and distal Kdo unit were investigated. The synthesis of 7-O-methyl and 7-azido-7-deoxy-7-epi-Kdo monosaccharide derivatives was achieved via an 8-O-TBS protected derivative, whereas methylation of O-7 at the proximal Kdo unit of the alpha-(2-->8)-linked Kdo disaccharide was conveniently accomplished via a 4,5; 4',5'; 7',8'-tri-O-carbonyl-protected disaccharide intermediate. Attempted epimerization at C-5 of the inner unit of a alpha-(2-->4)-linked Kdo disaccharide, however, resulted in formation of the corresponding 5,6-dehydro derivative, which was fully deprotected.

Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo.