Soluble Human Lectins at the Host-Microbe Interface.

Human lectins are integral to maintaining microbial homeostasis on the skin, in the blood, and at mucosal barriers. These proteins can recognize microbial glycans and inform the host about its microbial status. In accordance with their roles, their production can vary with tissue type. They also can have unique structural and biochemical properties, and they can influence microbial colonization at sites proximal and distal to their tissue of origin. In line with their classification as innate immune proteins, soluble lectins have long been studied in the context of acute infectious disease, but only recently have we begun to appreciate their roles in maintaining commensal microbial communities (i.e., the human microbiota). This review provides an overview of soluble lectins that operate at host-microbe interfaces, their glycan recognition properties, and their roles in physiological and pathological mechanisms.

Chemical glycobiology.

Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches are contributing great insight into the myriad biological functions of oligosaccharides.

Synthetic multivalent ligands in the exploration of cell-surface interactions.

Processes such as cell-cell recognition and the initiation of signal transduction often depend on the formation of multiple receptor-ligand complexes at the cell surface. Synthetic multivalent ligands are unique probes of these complex cell-surface-binding events. Multivalent ligands can be used as inhibitors of receptor-ligand interactions or as activators of signal transduction pathways. Emerging from these complementary applications is insight into how cells exploit multivalent interactions to bind with increased avidity and specificity and how cell-surface receptor organization influences signaling and the cellular responses that result.

Transforming the cell surface through proteolysis.

Protein shedding, or the proteolytic cleavage of a protein from the surface of a cell, is emerging as an important mechanism in the regulation of cellular activity but it is poorly understood. Growing evidence suggesting that protein shedding and protein function are closely linked may lead to new strategies for the treatment of a wide range of diseases.

Strength in numbers: non-natural polyvalent carbohydrate derivatives.

Many processes mediated by protein-carbohydrate interactions involve multivalent low-affinity binding, which is inherently difficult to study. New structural templates for the generation of multivalent carbohydrate displays have recently been developed, and tailored multivalent saccharide derivatives can now be used to study and modulate a wide variety of biological recognition events.

Tuning chemotactic responses with synthetic multivalent ligands.

BACKGROUND: Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses. Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency. How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined. The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function. Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response. RESULTS: To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization. We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis. The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency. Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane. CONCLUSIONS: Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria. This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both. Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses. Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses.

Synthesis of end-labeled multivalent ligands for exploring cell-surface-receptor-ligand interactions.

BACKGROUND: Ring-opening metathesis polymerization (ROMP) is a powerful synthetic method for generating unique materials. The functional group tolerance of ruthenium ROMP initiators allows the synthesis of a wide range of biologically active polymers. We generated multivalent ligands that inhibit cell surface L-selectin, a protein that mediates lymphocyte homing and leukocyte recruitment in inflammation. We hypothesized that these ligands function through specific, multivalent binding to L-selection. To examine this and to develop a general method for synthesizing multivalent materials with end-labels, we investigated functionalized enol ethers as capping agents in ruthenium-initiated ROMP. RESULTS: We synthesized a bifunctional molecule that introduces a unique end group by terminating ruthenium-initiated ROMP reactions. This agent contains an enol ether at one end and a masked carboxylic acid at the other. We conjugated a fluorescein derivative to an end-capped neoglycopolymer that had previously been shown to inhibit L-selection function. We used fluorescence microscopy to visualize neoglycopolymer binding to cells displaying L-selectin. Our results suggest that the neoglycopolymers bind specifically to cell surface L-selectin through multivalent interactions. CONCLUSIONS: Ruthenium-initiated ROMP can be used to generate biologically active, multivalent ligands terminated with a latent functional group. The functionalized polymers can be labeled with a variety of molecular tags, including fluorescent molecules, biotin, lipids or antibodies. The ability to conjugate reporter groups to ROMP polymers using this strategy has broad applications in the material and biological sciences.

Improved chemical synthesis of UDP-galactofuranose.

[reaction: see text] A reliable and efficient synthetic route to UDP-alpha-D-galactofuranose (UDP-Galf) has been developed. Reaction of UMP-N-methylimidazolide with Galf 1-phosphate proceeds rapidly to provide UDP-Galf with excellent reproducibility and in a yield approximately twice as high as those reported previously.

Staudinger ligation: a peptide from a thioester and azide.

[reaction: see text] The technique of native chemical ligation enables the total chemical synthesis of proteins. This method is limited, however, by an absolute requirement for a cysteine residue at the ligation juncture. Here, this restriction is overcome with a new chemical ligation method in which a phosphinobenzenethiol is used to link a thioester and azide. The product is an amide with no residual atoms.

High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide.

[figure: see text] The Staudinger ligation can be used to couple a peptide with a C-terminal phosphinothioester to another with an N-terminal alpha-azido group to form a single peptide that contains no residual atoms. Here diphenylphosphinomethanethiol thioesters are shown to give high isolated yields for this transformation. This finding provides precedent for a powerful and versatile new method for the total synthesis of proteins.

Synthesis of cyclic sulfates by halocyclization.

[reaction: see text]. We report the synthesis of cyclic sulfates by halocyclization. The resulting cyclic sulfate products can be opened selectively with sodium azide to transform them into highly functionalized compounds that contain azide, alcohol, and halide groups.

Solution conformation of Lewis a--derived selectin ligands is unaffected by anionic substituents at the 3'- and 6'-positions.

The selectins are a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium. E-, P-, and L-selectin recognize various derivatives of the Lewis(a) and Lewis(x) trisaccharides. The distribution of negative charges on the Lewis(a) and Lewis(x) oligosaccharides appears to be an important factor in their binding by the selectins. Previous work exploring this electrostatic dependence found that a series of synthetic anionic trisaccharides, 3'-sulfo, 3'-phospho, 6'-sulfo, and 3',6'-disulfo Lewis(a)-(Glc), exhibited differing selectin inhibitory efficacies. To explore the possibility that these differences arise from conformational differences between the sugars, the solution structures of these trisaccharides were determined using NMR and molecular dynamics simulations. Interproton distances and interglycosidic torsion angles were determined at 37 degrees C using NOESY buildup curves and 1D LRJ experiments, respectively. Data from both experiments agreed well with predictions made from 2000 picosecond unrestrained molecular dynamics simulations. We found that 3'-sulfation did not alter the core Lewis(a) conformation, a finding that reaffirms the results of previous study. In addition, we found that sulfation at the 6' position also leaves the trisaccharide conformation unperturbed. This is significant because the proximity of the 6'-sulfate group to the fucose ring might have altered the canonical Lewis (a) structure. The disulfate exhibited greater flexibility than the other derivatives in dynamics simulations, but not so much as to affect NOE and heteronuclear coupling constant measurements. Taken together, our findings support the use of Lewis(a) as a template onto which charged groups may be added without significantly altering the trisaccharide's structure.

Model of the interactions of calichemicin gamma 1 with a DNA fragment from pBR322.

An analysis of the binding interactions of several DNA-drug complexes that utilize carbohydrates for DNA recognition has been undertaken. It is proposed that the carbohydrate residues function as general minor groove binding elements, and the stereochemistry of aglycone attachment sites is generally disposed to promote a right-handed helical geometry that is complementary to right-handed DNA. The constitution and stereochemistry of the DNA double-strand cleaving agent calichemicin gamma 1 is consistent with this analysis. Docking experiments with computer-generated models of this drug and a dodecamer duplex that was found to serve as a calichemicin cleavage site were performed to gain insight into the origin of the drug's sequence-selective binding and cutting properties. A model is presented that provides a molecular level understanding of the double-strand cleavage patterns that result from the action of calichemicin gamma 1 on DNA.

Glycoprotein-inspired materials promote the proteolytic release of cell surface L-selectin.

The proteolytic release, or shedding, of a cell surface protein can serve a regulatory role; the process liberates a soluble form of the protein into circulation while downregulating its cell surface concentration. The characteristics that render a protein susceptible to proteolytic cleavage are not known. We hypothesized that the clustering of a protein at the cell surface might target it for proteolysis. To test this hypothesis, we synthesized molecules that display multiple copies of sulfated galactose residues, termed neoglycopolymers, that are designed to mimic natural ligands for the cell adhesion protein L-selectin. We found that treatment of human neutrophils with the neoglycopolymers resulted in a dose-dependent loss of L-selectin from the cell surface, while monovalent compounds and unsulfated neoglycopolymers had no effect. Because L-selectin is an important mediator in the inflammatory response, such compounds could lead to novel antiinflammatory drugs. Moreover, molecules that control receptor shedding can be used to alter cellular responsiveness to specific ligands or to promote responses at distal sites; consequently, these results have broad implications for regulating the location and presentation of important biomolecules.

Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving.

Nearest neighbor interactions affect the stabilities of triple-helical complexes. Within a pyrimidine triple-helical motif, the relative stabilities of natural base triplets T.AT, C + GC, and G.TA, as well as triplets, D3.TA and D3.CG, containing the nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3) were characterized by the affinity cleaving method in the context of different flanking triplets (T.AT, T.AT: T.AT, C + GC: C + GC, T.AT: G + GC, C + GC). The to be insensitive to substitutions in either the 3' or 5' directions, while the relative stabilities of triple helices containing C + GC triplets decreased as the number of adjacent C + GC triplets increased. Triple helices incorporating a G.TA interaction were most stable when this triplet was flanked by two T.AT triplets and were adversely affected when a C + GC triplet was placed in the adjacent 5' direction. Similarly, complexes containing D3.TA or D3.CG triplets were most stable when the triplet was flanked by two T.AT triplets but were destabilized when the adjacent 3' neighbor position was occupied with a C + GC triplet. This information regarding sequence composition effects in triple-helix formation establishes a set of guidelines for targeting sequences of double-helical DNA by the pyrimidine triple-helix motif.

Motility and chemotaxis of filamentous cells of Escherichia coli.

Filamentous cells of Escherichia coli can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal E. coli organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous E. coli are discussed.

A strategy for designing inhibitors of beta-amyloid toxicity.

beta-Amyloid peptide is the major protein component of Alzheimer's plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of beta-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with beta-amyloid, is combined with a disrupting element, which alters beta-amyloid aggregation pathways. The synthesis, biophysical characterization, and biological activity of such an inhibitor is reported. This prototype inhibitor is composed of residues 15-25 of beta-amyloid peptide, designed to function as the recognition element, linked to an oligolysine disrupting element. The inhibitor does not alter the apparent secondary structure of beta-amyloid nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. Evidence for these effects includes changes in fibril morphology and a reduction in thioflavin T fluorescence. In addition to its influence on the physical properties of beta-amyloid aggregates, the inhibitor completely blocks beta-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of beta-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.

Specificity of C-glycoside complexation by mannose/glucose specific lectins.

The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C-glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed.

Structure-function relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF.

Beta-amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity. These compounds contain a recognition element, designed to bind to Abeta, linked to a disrupting element, designed to interfere with Abeta aggregation. On the basis of this strategy, a hybrid peptide was synthesized with the sequence KLVFF (residues 16-20 of Abeta) as the recognition element and a lysine hexamer as the disrupting element; this compound protects cells in vitro from Abeta toxicity [Pallitto, M. M., et al. (1999) Biochemistry 38, 3570]. To determine if the length of the disrupting element could be reduced, peptides were synthesized that contained the KLVFF recognition element and a sequence of one to six lysines as disrupting elements. All compounds enhanced the rate of aggregation of Abeta, with the magnitude of the effect increasing as the number of lysines in the disrupting element increased. The greatest level of protection against Abeta toxicity was achieved with compounds containing disrupting elements of three or more lysines in sequence. A peptide with an anionic disrupting element, KLVFFEEEE, had activity similar to that of KLVFFKKKK, in both cellular toxicity and biophysical assays, whereas a peptide with a neutral polar disrupting element, KLVFFSSSS, was ineffective. Protective compounds retained activity even at an inhibitor:Abeta molar ratio of 1:100, making these some of the most effective inhibitors of Abeta toxicity reported to date. These results provide critical insight needed to design more potent inhibitors of Abeta toxicity and to elucidate their mechanism of action.

Recognition sequence design for peptidyl modulators of beta-amyloid aggregation and toxicity.

beta-Amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity, based on linking a recognition element for Abeta to a disrupting element designed to interfere with Abeta aggregation. One such compound, with the 15-25 sequence of Abeta as the recognition element and a lysine hexamer as the disrupting element, altered Abeta aggregation kinetics and protected cells from Abeta toxicity [Ghanta et al. (1996) J. Biol. Chem. 271, 29525]. To optimize the recognition element, peptides of 4-8 residues composed of overlapping sequences within the 15-25 domain were synthesized, along with hybrid compounds containing those recognition sequences coupled to a lysine hexamer. None of the recognition peptides altered Abeta aggregation kinetics and only two, KLVFF and KLVF, had any protective effect against Abeta toxicity. The hybrid peptide KLVFF-KKKKKK dramatically altered Abeta aggregation kinetics and aggregate morphology and provided significantly improved protection against Abeta toxicity compared to the recognition peptide alone. In contrast, FAEDVG-KKKKKK possessed only modest inhibitory activity and had no marked effect on Abeta aggregation. The scrambled sequence VLFKF was nearly as effective a recognition domain as KLVFF, suggesting the hydrophobic characteristics of the recognition sequence are critical. None of the cytoprotective peptides prevented Abeta aggregation; rather, they increased aggregate size and altered aggregate morphology. These results suggest that coupling recognition with disrupting elements is an effective generalizable strategy for the creation of Abeta inhibitors. Significantly, prevention of Abeta aggregation may not be required for prevention of toxicity.