David R. Bundle

Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands

The diseases caused by Shiga and cholera toxins account for the loss of millions of lives each year. Both belong to the clinically significant subset of bacterial AB5 toxins consisting of an enzymatically active A subunit that gains entry to susceptible mammalian cells after oligosaccharide recognition by the B5 homopentamer. Therapies might target the obligatory oligosaccharide–toxin recognition event, but the low intrinsic affinity of carbohydrate–protein interactions hampers the development of low-molecular-weight inhibitors. The toxins circumvent low affinity by binding simultaneously to five or more cell-surface carbohydrates. Here we demonstrate the use of the crystal structure of the B5 subunit of Escherichia coli O157:H7 Shiga-like toxin I (SLT-I) in complex with an analogue of its carbohydrate receptor to design an oligovalent, water-soluble carbohydrate ligand (named STARFISH), with subnanomolar inhibitory activity. The in vitro inhibitory activity is 1–10-million-fold higher than that of univalent ligands and is by far the highest molar activity of any inhibitor yet reported for Shiga-like toxins I and II. Crystallography of the STARFISH/Shiga-like toxin I complex explains this activity. Two trisaccharide receptors at the tips of each of five spacer arms simultaneously engage all five B subunits of two toxin molecules.

Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis

The first fully synthetic glycopeptide vaccines against a fungal disease have been used to combat disseminated candidiasis in mice. Six T cell peptides found in Candida albicans cell wall proteins were selected by algorithm peptide epitope searches; each was synthesized and conjugated to the fungal cell wall β-mannan trisaccharide [β-(Man)3] by novel saccharide-peptide linker chemistry to create glycopeptide conjugates. The six proteins were selected because of expression during human candidiasis and cell wall association and included: fructose-bisphosphate aldolase (Fba); methyltetrahydropteroyltriglutamate (Met6); hyphal wall protein-1 (Hwp1); enolase (Enol); glyceraldehyde-3-phosphate dehydrogenase (Gap1); and phosphoglycerate kinase (Pgk1). By immunization protocols favoring production of protective antibody, the β-(Man)3-Fba, β-(Man)3-Met6 and β-(Man)3-Hwp1 induced protection evidenced by survival and reduced kidney fungal burden, the β-(Man)3-Eno1 and β-(Man)3-Gap1 gave moderate protection, and the β-(Man)3-Pgk1 slightly enhanced disease. For the β-(Man)3-Fba conjugate, protection was uniquely acquired through immunity against the carbohydrate and the Fba peptide. This approach based on fully synthetic chemically defined immunogens should be generally useful in vaccine development.

Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (k ≈ 0.25 s). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 × 105M s. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the on-rate was the same as that obtained with Fab and scFv monomer.

Linear PADRE T Helper Epitope and Carbohydrate B Cell Epitope Conjugates Induce Specific High Titer IgG Antibody Responses

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

Micro‐Scale Frontal Affinity Chromatography with Mass Spectrometric Detection: A New Method for the Screening of Compound Libraries

In one run the binding constants Kd for all the active components of a ligand library at sub-microgram quantities can be determined. A mixture of ligands is continuously infused through a column of immobilized receptor, and the eluent analyzed by electrospray mass spectrometry. From the affinity chromatogram produced (see picture) the breakthrough volume of a single compound and hence its Kd value can be determined.

Sequential Interchange of Four Amino Acids from Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 → Gly, Gly-235 → Ser, Leu-266 → Met, and Gly-268 → Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with thek cat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 → Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the k cat and increased specificity. The increases observed in k cat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

A Mutational Analysis of the Globotriaosylceramide-binding Sites of Verotoxin VT1

Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb3 receptors which present the Pktrisaccharide Galα(1−4)Galβ(1–4)Glcβ. Crystallographic studies have identified three Pk trisaccharide (Pk-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of Pk-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to Pk-glycoside and pentavalent Pk trisaccharide (pentaSTARFISH) in solution and Gb3 presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb3 involved all three sites. Binding of all single site mutants to liposomal Gb3 was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb3 in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb3, even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb3. Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb3 trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.

Antigenic determinants of Salmonella serogroups A and D1.Synthesis of trisaccharide glycosides for use as artificial antigens

The disaccharide glycoside 8-methoxycarbonyloctyl 4,6-O-cyclohexylidine-2-O-(tetra-O-benzyl-alpha-D-galactopyranosyl)-alpha-D-manno pyranoside (7) was used as a common intermediate to the trisaccharide determinants of both Salmonella serogroups A and D1. Acetalation of 8-methoxycarbonyloctyl alpha-D-mannopyranoside provided the 4,6-acetal derivative, which was selectively benzoylated to give the partially protected mannoside 4. Reaction of 4 with tetra-O-benzyl-alpha-D-galacto-pyranosyl chloride afforded the fully protected disaccharide, which, after transesterification, gave the selectively blocked, disaccharide glycoside (7). Addition of tyvelose by way of its 2,4-di-O-benzoyl-3,6-dideoxy-alpha-D-arabino-hexopyranosyl chloride derivative gave the blocked trisacchride determinant of Salmonella serogroup D1. 2,4,-Di-O-benzyl-3,6-dideoxy-alpha-D-ribo-hexopyranosyl chloride reacted with 7 to provide, after removal of blocking groups, the paratose-containing determinant of serogroup A.

Structural basis of peptide–carbohydrate mimicry in an antibody-combining site

The structure of a complex between the Fab fragment of the antibody (SYA/J6) specific for the cell surface O-antigen polysaccharide of the pathogen Shigella flexneri Y and an octapeptide (Met–Asp–Trp–Asn–Met–His–Ala–Ala), a functional mimic of the O-antigen, has been determined at 1.8-Å resolution. Comparison of the structure with that of the complex with the pentasaccharide antigen [→2)-α-l-Rha-(1→2)-α-l-Rha-(1→3)-α-l-Rha-(1→3)-β-d-GlcNAc-(1→2)-α-l-Rha-(1→] reveals the molecular recognition process by which a peptide mimics a carbohydrate in binding to an antibody. The binding modes of the two ligands differ considerably. Octapeptide binding complements the shape of the combining site groove much better than pentasaccharide binding. Moreover, the peptide makes a much greater number of contacts (126), which are mostly van der Waals interactions, with the Fab than the saccharide (74). An unusual feature is also the involvement of 12 water molecules in mediating hydrogen bonds between residues within the peptide or of the peptide and Fab. Despite better shape complementarity and greater number of contacts, the octapeptide binds with an affinity (KA = 2.5 × 105 M-1, measured by calorimetry) only ≈2-fold tighter than the pentasaccharide. The structural results are relevant to the design of peptide mimetics with improved affinity for use as vaccines.

Synthesis of antigenic determinants of the Brucella a antigen, utilizing methyl 4-azido-4,6-dideoxy-α-D-mannopyranoside efficiently derived from D-mannose

A strategy for the synthesis of Brucella O-antigenic determinants containing 2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues is described. The approach adopted also permits the N-acyl moiety to be varied. A high-yield synthesis of methyl 4-azido-4,6-dideoxy-alpha-D-mannopyranoside from D-mannose on the 10-20-g scale provided the key intermediate. Regioselective acetylation of 7 gave the 2-acetate 8, which, on treatment with benzyl trichloroacetimidate, provided methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside. This compound served as a common precursor to the glycosyl donor 12 and acceptor 10 molecules. Silver trifluoromethanesulphonate-promoted glycosylation of 10 by 12 gave a disaccharide derivative, hydrogenolysis of which gave methyl 4-amino-2-O-(4-amino-4,6-dideoxy-alpha-D-mannopyranosyl)-4,6-dideoxy- alpha-D-mannopyranoside from which the N-formyl and N-acetyl derivatives were obtained. Deacetylation of 13 followed by glycosylation with 12 gave a trisaccharide derivative. The N-formylated disaccharide 17 inhibited the binding of Brucella O-polysaccharide to Brucella-specific monoclonal antibodies.