Biomedical subjects

G D Armstrong

Publications and source records attributed to G D Armstrong.

At least 19 recordsLinked to original sources

PubMed1998

Structure of the shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3.

Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans. Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer). The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin. Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide. The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues. All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane. The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.

Bacterial Toxins↗

PubMed1998

Modeling the carbohydrate-binding specificity of pig edema toxin.

The wild-type binding pentamer of Shiga-like toxin IIe (SLT-IIe) binds both the globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) cell surface glycolipids, whereas the double mutant GT3 (Q65E/K67Q) exhibits a marked preference for Gb3 [Tyrrell, G. J., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 524-528]. We modeled three unique sites (sites 1-3) for binding of the carbohydrate moiety of Gb3 to GT3 and SLT-IIe, on the basis of the three sites observed for the SLT-I pentamer [Ling, H., et al. (1998) Biochemistry 37, 1777-1788]. Examination of the three sites in light of various mutation and binding data strongly suggested that one of the binding sites plays a role in the change of specificity observed for the GT3 mutant. We applied several modeling techniques, and developed a model for binding of the carbohydrate moiety of Gb4 to this site of the SLT-IIe binding pentamer. This model is consistent with a wide variety of mutation and binding data and clearly shows the importance of the terminal GalNAc residue of Gb4, as well as that of the two mutated residues of GT3, to the intermolecular interaction.

Amino Acid Sequence↗

PubMed1997

Effect of carbon source on localized adherence of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains attach to epithelial cells as discrete clusters of bacteria which are localized at a few sites on the cell surface. Previously, it was shown that this localized-adherence (LA) phenotype is induced by specific growth conditions. We found that wild-type EPEC attached to HEp-2 cells in an LA pattern when the bacteria were grown in Dulbecco's modified Eagle medium (DMEM) containing glucose as the carbon source. In contrast, bacteria incubated in DMEM containing galactose did not adhere to epithelial cells. The latter results were similar to those observed when JPN15, an LA-negative strain, was grown under conditions which promoted bacterial binding. The differences in attachment of wild-type EPEC were independent of the stage of log-phase growth of the cultures and of the number of CFU incubated with the HEp-2 monolayers. Expression of the adherence phenotype by organisms grown in glucose was associated with increased expression of intimin and bundle-forming pilin. In contrast, bacteria grown in medium containing galactose expressed these proteins at levels similar to those observed when JPN15 was grown in medium containing glucose.

Bacterial Adhesion↗

PubMed1996

Interactions between Campylobacter jejuni and lipids.

We previously showed that motility plays several key roles in Campylobacter jejuni pathogenesis, including increasing the efficiency of C. jejuni attachment to host epithelial cells. To further characterize C. jejuni attachment, we first examined the role of carbohydrates. Experiments with Chinese hamster ovary (CHO) cell mutants with defined defects in complex carbohydrate biosynthesis revealed that oligosaccharide sequences probably play a subordinate role in C. jejuni attachment to eukaryotic cells. Simple sugars such as mannose, fucose, glucose, N-acetylglucosamine, maltose, and galactose also did not significantly alter the binding of C. jejuni to CHO cells. Thin-layer chromatography overlay analysis with lipids extracted from CHO cells suggested that C. jejuni binds to lipids. Lipid binding was further investigated using a receptor-based enzyme-linked immunosorbent assay. Hydrophobic interactions were determined to play a minor role in binding, since tetramethylurea, a strong inhibitor of hydrophobic interactions, did not significantly decrease binding between C. jejuni and lipids. The interaction was dissected further by comparing the binding of C. jejuni to lipids and their derivatives. The results showed that binding was greatest to the entire lipid structure and decreased in affinity when portions of the lipid were removed. Thin-layer chromatography overlay analysis showed that lipids with unsaturated fatty acids were bound with the highest affinity. Our results suggest that C. jejuni may interact with lipids in host cell membranes. However, lipids only partially inhibited C. jejuni binding to CHO cells, suggesting that multiple interactions occur between the bacteria and host cells.

Animals↗

PubMed1995

Utilization of sialic acid-binding synthetic peptide sequences derived from pertussis toxin as novel anti-inflammatory agents.

Pertussis toxin, a virulence factor produced by the organism Bordetella pertussis, has been shown to have functional similarities with selectins and to bind to similar sialic acid-containing oligosaccharides structures. Previously, we demonstrated that the amino-terminal region of the S2 subunit of pertussis toxin contained a short six amino acid sequence (SPYGRC) which displayed reasonable homology to a sequence that constitutes a portion of the sialic acid binding site in wheat germ agglutinin. Synthetic peptides containing this hexapeptide motif had the ability to bind to sialic acid-containing glycoconjugates including the putative oligosaccharide receptors (sialyl Lewis X and sialyl Lewis A) for selectins. Control peptides containing randomized sequences were inactive at inhibiting binding, indicating that the hexapeptide motif is important for interacting with sialic acid. Since pertussis toxin-derived peptides demonstrated the ability to interact with selectin receptors, we speculated that they should antagonize selectin-mediated inflammatory activity. To test this hypothesis, we evaluated the peptides for the ability to reduce neutrophil binding to activated endothelial cells as well as the anti-inflammatory activity in the mouse footpad swelling assay. Both S2 peptides were active at reducing neutrophil binding and footpad swelling, while the randomized control peptides were inactive.

Amino Acid Sequence↗

PubMed1995

A phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pk-trisaccharide receptors attached to chromosorb for preventing hemolytic-uremic syndrome.

A double-blind, placebo-controlled study was conducted to document possible side effects associated with oral consumption of synthetic verotoxin (VT, shiga-like toxin) Pk-trisaccharide receptor sequences attached to Chromosorb (Synsorb-Pk) by healthy adult volunteers. Synsorb-Pk reclaimed from volunteer stool samples was also analyzed to determine if its VT-binding activity was affected by exposure to the pH extremes and digestive processes of the human gastrointestinal tract. No participant reported any Synsorb-Pk-related adverse reactions, and no clinically important trends in laboratory data were evident. Synsorb-Pk recovered from stools retained its ability to absorb VT in polymyxin extracts of VT-producing Escherichia coli and also neutralized VT when mixed in vitro with VT-positive stools from children with hemorrhagic colitis or hemolytic-uremic syndrome (HUS). These results suggest a potential use for Synsorb-Pk in preventing HUS in patients infected with VT-producing E. coli.

Absorption↗

PubMed1995

Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells.

Enteropathogenic Escherichia coli (EPEC) O111:H2, O119:H6, or O142:H6 caused rapid detachment of Chinese hamster ovary (CHO) cell monolayers within 2 to 4 h of cocultivation. CHO cell detachment was not promoted by nonenteropathogenic E. coli (O125:H4, O126:H27, O157:H7, and O26:H11) and could not be attributed to EPEC production of enterohemolysin or Shiga-like toxins. In contrast, EPEC strains did not promote rapid detachment of Lec1, Lec2, or Lec8 CHO cell monolayers. These CHO cell Lec mutants all express abbreviated glycan sequences on membrane glycoproteins and glycolipids. Although EPEC strains failed to alter the adherent properties of Lec2 cells lacking only terminal sialic acid groups, EPEC adherence to the Lec2 mutant was indistinguishable from that observed with wild-type CHO cells. There was also no significant difference in EPEC-induced actin accumulation or invasion of Lec2 cells. In contrast, EPEC localized adherence to Lec1 and Lec8 mutants, lacking sialyllactosamine (Lec1) or sialic acid and galactose (Lec8) sequences, was reduced by 84 and 93%, respectively. Our results suggest that lactosamine sequences [beta Gal(1-4 or 1-3)beta GlcNAc] not containing sialic acid are sufficient for EPEC adherence, actin accumulation, and invasion of CHO cells. Sialic acid groups, however, may be necessary for EPEC-mediated CHO cell detachment.

Animals↗

PubMed1995

Campylobacter jejuni motility and invasion of Caco-2 cells.

We investigated the influence of motility on Campylobacter jejuni binding and invasion of Caco-2 cells. C. jejuni was motile in soft agar at basic (pH 8.5) and neutral pH values representative of the intestinal environment. However, C. jejuni was immobilized at pH 5.0. The inability of C. jejuni to swarm on soft agar at pH 5.0 was not related to flagellar depolymerization or loss of viability. In tissue culture medium, C. jejuni displayed typical periods of straight swimming punctuated by tumbling behavior. This behavior was altered when the viscosity of the medium was adjusted to mimic the viscosity of intestinal mucus. C. jejuni showed longer periods of straight swimming with significantly increased velocity followed by pauses instead of tumbles. The binding and invasion of C. jejuni in Caco-2 cells also increased significantly in high-viscosity growth medium. We speculate that the swimming behavior of C. jejuni in a viscous environment may be an important factor in the interaction of these organisms with host epithelial cells. The pH, which affects C. jejuni motility, may also influence the tropism of these organisms.

Bacterial Adhesion↗

PubMed1994

The crystal structure of pertussis toxin.

BACKGROUND: Pertussis toxin is an exotoxin of the A-B class produced by Bordetella pertussis. The holotoxin comprises 952 residues forming six subunits (five different sequences, S1-S5). It plays an important role in the development of protective immunity to whooping cough, and is an essential component of new acellular vaccines. It is also widely used as a biochemical tool to ADP-ribosylate GTP-binding proteins in the study of signal transduction. RESULTS: The crystal structure of pertussis toxin has been determined at 2.9 A resolution. The catalytic A-subunit (S1) shares structural homology with other ADP-ribosylating bacterial toxins, although differences in the carboxy-terminal portion explain its unique activation mechanism. Despite its heterogeneous subunit composition, the structure of the cell-binding B-oligomer (S2, S3, two copies of S4, and S5) resembles the symmetrical B-pentamers of the cholera toxin and Shiga toxin families, but it interacts differently with the A-subunit. The structural similarity is all the more surprising given that there is almost no sequence homology between B-subunits of the different toxins. Two peripheral domains that are unique to the pertussis toxin B-oligomer show unexpected structural homology with a calcium-dependent eukaryotic lectin, and reveal possible receptor-binding sites. CONCLUSION: The structure provides insight into the pathogenic mechanisms of pertussis toxin and the evolution of bacterial toxins. Knowledge of the tertiary structure of the active site forms a rational basis for elimination of catalytic activity in recombinant molecules for vaccine use.

Amino Acid Sequence↗

PubMed1994

Structure of a pertussis toxin-sugar complex as a model for receptor binding.

Pertussis toxin is an exotoxin from the bacterium Bordetella pertussis which is important the pathogenesis of whooping cough and the generation of a protective immune response. The diverse biological activities of the toxin depend on its ability to recognize carbohydrate-containing receptors on a wide variety of eukaryotic cells. We present here the crystal structure of pertussis toxin complexed with a soluble oligosaccharide from transferrin. Binding sites for the terminal sialic acid-galactose moiety are revealed on both subunits S2 and S3 of the B-oligomer. Identification of amino acid residues involved in receptor binding will improve the design of genetically inactivated toxins for use in new acellular whooping cough vaccines.

Bacterial Vaccines↗

PubMed1994

Oligosaccharide sequences attached to an inert support (SYNSORB) as potential therapy for antibiotic-associated diarrhea and pseudomembranous colitis.

Toxin A produced by Clostridium difficile, the causative agent of pseudomembranous colitis and antibiotic-associated diarrhea, was shown to bind to synthetic oligosaccharide sequences attached to an inert support (SYNSORB). The oligosaccharide sequences that bind to toxin A were related to sequences previously identified as potential receptors for the toxin. Various SYNSORBs containing a variety of oligosaccharides were examined for their potential to neutralize toxin A activity from toxin-containing solutions as well as clinical stool samples from patients with either pseudomembranous colitis or antibiotic-associated diarrhea. The results from neutralization experiments suggest SYNSORB can effectively neutralize toxin A activity from stool samples and thus could serve as a potential therapy for C. difficile-associated diarrhea.

Animals↗

PubMed1994

The 70-kilodalton pertussis toxin-binding protein in Jurkat cells.

125I-ASD photoaffinity-labeling derivatives of pertussis toxin (125I-ASD-PT) or lipopolysaccharide (125I-ASD-LPS) labeled similar 70-kDa proteins in Jurkat cells, a cell line derived from human CD4+ T lymphocytes. Labeling of this 70-kDa protein by 125I-ASD-PT was inhibited by underivatized PT but not by underivatized LPS. However, an immunoglobulin M monoclonal antibody with specificity for the p73 LPS receptor in murine splenocytes (S. W. Bright, T.-Y. Chen, L. M. Flebbe, M.-G. Lei, and D. C. Morrison, J. Immunol. 145:1-7, 1990) inhibited 125I-ASD-PT labeling of the 70-kDa species in Jurkat cells. Our results suggested that PT may bind to the same 70-kDa protein as LPS does in Jurkat cells but that PT and LPS bind to different sites on this receptor candidate. 125I-ASD-PT photoaffinity labeling of the 70-kDa protein was also inhibited by underivatized glycoproteins to which PT has been shown to bind, and this inhibition correlated with the relative binding affinities of the glycoproteins for PT. 125I-ASD derivatives of two sialic acid-specific plant lectins, Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin, with oligosaccharide binding specificities similar to those of PT also labeled a 70-kDa protein in Jurkat cells. This suggests that the 70-kDa PT receptor candidate in Jurkat cells likely contains sialooligosaccharide sequences to which PT, M. amurensis leukoagglutinin, and S. nigra agglutinin bind. The cross-reacting epitope recognized by monoclonal antibody 5D3 in this 70-kDa species might overlap the PT- and LPS-binding sites.

CD4-Positive T-Lymphocytes↗

PubMed1993

Hydrophobic binding of pertussis toxin is enhanced by oligosaccharide receptors.

Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, the etiologic agent of whooping cough. Pertussis toxin is an oligomeric A-B class toxin composed of an ADP-ribosyltransferase S1 (A) subunit and a B oligomer containing lectin-like binding domains. The carbohydrate binding specificity of the B oligomer is for sialooligosaccharide sequences expressed on target cell receptors and asparagine-linked glycans found in many serum glycoproteins. Pertussis toxin also has the ability to bind to the inert surfaces of culture tubes. In this report we present data showing that pertussis toxin binding to polypropylene microcentrifuge tubes was enhanced in a time- and concentration-dependent manner by the addition of soluble glycoprotein or oligosaccharide receptor analogs. Evidence obtained using the hydrophilic and hydrophobic surfaces of Gel Bond electrophoresis casting film indicated that receptor-enhanced binding was likely due to hydrophobic interactions. Hydrophobic binding of the isolated B oligomer of pertussis toxin was enhanced only in the presence of high concentrations of glycoproteins. Therefore, the S1 (A) subunit of pertussis holotoxin appears to play a role in receptor-enhanced hydrophobic binding. We propose, therefore, that pertussis toxin binding to its receptors may expose or preferentially orient hydrophobic residues that may contribute to the functional association of the toxin with host cell plasma membranes and delivery of the S1 subunit to its intracellular target.

Cell Membrane↗

PubMed1992

Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.

Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.

Amino Acid Sequence↗

PubMed1991

Synthesis and characterization of a Pertussis toxin-biotin conjugate.

We prepared a Pertussis toxin-biotin conjugate and found its biological properties to be similar to those of native Pertussis toxin with respect to the hemagglutination, Chinese hamster ovary cell, and lymphocyte proliferation assays. Direct binding to Chinese hamster ovary and Jurkat cells was observed using fluorescence microscopy. Pertussis toxin-biotin was also found to possess similar glycoconjugate binding specificities as those of 125I-labeled Pertussis toxin.

Animals↗

PubMed1991

High-level production of murine interleukin-5 (IL-5) utilizing recombinant baculovirus expression. Purification of the rIL-5 and its use in assessing the biologic role of IL-5 glycosylation.

We describe here a recombinant baculovirus expression system useful for high level production of murine recombinant interleukin-5 (rIL-5). In addition, we describe a single-step technique of purification of the rIL-5 from the baculovirus-infected Sf9 cell supernatants, using an anti-IL-5 affinity column. The baculovirus-derived rIL-5 has physical properties and functional activities in various lymphoid cell assays similar to those of natural T cell-derived IL-5 and reacts with anti-IL-5 antibodies. Finally, the rIL-5 is similar to natural T cell-derived IL-5 in manifesting heterogeneous glycosylation; however, glycosylation does not appear to be necessary for biologic function, at least in a lymphoid cell proliferation assay.

Animals↗

PubMed1991

Investigation of Shiga-like toxin binding to chemically synthesized oligosaccharide sequences.

Shiga-like toxin (SLT)-I and SLT-II/IIc bound to Synsorbs containing synthetic alpha Gal(1-4)beta Gal (P1 disaccharide), alpha Gal(1-4)beta GlcNAc (P1 trisaccharide), or alpha Gal(1-4)beta Gal(1-4)beta Glc (Pk trisaccharide) sequences but not to Synsorbs containing alpha Gal(1-3)beta Gal, alpha Gal(1-3)beta Gal(1-4)beta GlcNAc, or the hydrophobic oligosaccharide linkage arm. SLT-I had a preference for Synsorbs containing trisaccharides, whereas SLT-II/IIc binding was less selective. 125I-labeled SLT-I remained bound to Pk trisaccharide Synsorb in the presence of lactose, galactose, or EDTA but was partially released by acetic acid, guanidine HCl, or a 10% solution of SDS. Vero cells coincubated with Pk trisaccharide Synsorb and SLT I extract were protected from this toxin, whereas Pk trisaccharide Synsorb was much less efficient at neutralizing SLT-II/IIc activity in Vero cell coincubation experiments. The SLT-IIc component was not responsible for the inefficient neutralization. Results suggest that synthetic oligosaccharide sequences related to the P blood group antigens coupled to inert matrices could be useful for rapid diagnosis or possibly therapeutic intervention in enterohemorrhagic Escherichia coli infections.

Animals↗

PubMed1990

Comparison of the lectin-like activity of pertussis toxin with two plant lectins that have differential specificities for alpha (2-6) and alpha (2-3)-linked sialic acid.

In this report we have compared the lectin-like properties of Pertussis toxin with two plant lectins which are known to possess different specificities towards terminal Neu5Ac Gal linkages on glycoconjugates. The hemagglutinin from elderberry bark (Sambucus nigra) has a binding specificity for terminal Neu5Ac alpha (2-6) Gal sequences and was found to bind a series of glycoconjugates with a similar specificity as Pertussis toxin. The binding specificity of Pertussis toxin was different from that of the leukoagglutinin from the seeds of Maackia amurensis which preferentially binds terminal Neu5Ac alpha (2-3) Gal sequences. These observations confirm the specificity of Pertussis toxin for Neu5Ac alpha (2-6) Gal glycoconjugate sequences.

Binding, Competitive↗