Laura L. Kiessling

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.

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.

Inter-receptor communication through arrays of bacterial chemoreceptors

The sensing mechanisms of chemotactic bacteria allow them to respond sensitively to stimuli. Escherichia coli, for example, respond to changes in chemoattractant concentration of less than 10% over a range spanning six orders of magnitude. Sensitivity over this range depends on a nonlinear relationship between ligand concentration and output response. At low ligand concentrations, substantial amplification of the chemotactic signal is required; however, the mechanism responsible for this amplification remains unclear. Here we demonstrate that inter-receptor communication within a lattice acts to amplify and integrate sensory information. Synthetic multivalent ligands that interact through the low-abundance, galactose-sensing receptor Trg stabilize large clusters of chemoreceptors and markedly enhance signal output from these enforced clusters. On treatment with multivalent ligands, the response to the attractant serine is amplified by at least 100-fold. This amplification requires a full complement of chemoreceptors; deletion of the aspartate (Tar) or dipeptide (Tap) receptors diminishes the amplification of the serine response. These results demonstrate that the entire array is involved in sensing. This mode of information exchange has general implications for the processing of signals by cellular receptors.

A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.

A strategy for designing inhibitors of beta-amyloid toxicity.

β-Amyloid peptide is the major protein component of Alzheimers plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of β-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with β-amyloid, is combined with a disrupting element, which alters β-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 β-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 β-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 β-amyloid aggregates, the inhibitor completely blocks β-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of β-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.

Chemical approaches to glycobiology.

Glycans are ubiquitous components of all organisms. Efforts to elucidate glycan function and to understand how they are assembled and disassembled can reap benefits in fields ranging from bioenergy to human medicine. Significant advances in our knowledge of glycan biosynthesis and function are emerging, and chemical biology approaches are accelerating the pace of discovery. Novel strategies for assembling oligosaccharides, glycoproteins, and other glycoconjugates are providing access to critical materials for interrogating glycan function. Chemoselective reactions that facilitate the synthesis of glycan-substituted imaging agents, arrays, and materials are yielding compounds to interrogate and perturb glycan function and dysfunction. To complement these advances, small molecules are being generated that inhibit key glycan-binding proteins or biosynthetic enzymes. These examples illustrate how chemical glycobiology is providing new insight into the functional roles of glycans and new opportunities to interfere with or exploit these roles.

Glycopolymer probes of signal transduction

Glycans are key participants in biological processes ranging from reproduction to cellular communication to infection. Revealing glycan roles and the underlying molecular mechanisms by which glycans manifest their function requires access to glycan derivatives that vary systematically. To this end, glycopolymers (polymers bearing pendant carbohydrates) have emerged as valuable glycan analogs. Because glycopolymers can readily be synthesized, their overall shape can be varied, and they can be altered systematically to dissect the structural features that underpin their activities. This review provides examples in which glycopolymers have been used to effect carbohydrate-mediated signal transduction. Our objective is to illustrate how these powerful tools can reveal the molecular mechanisms that underlie carbohydrate-mediated signal transduction.

High-Throughput Discovery of Synthetic Surfaces That Support Proliferation of Pluripotent Cells

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors, the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors, however, may be needed to guide cell growth and differentiation. To provide access to such new materials, we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait, approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested, and seven cell-binding peptides were identified. Of these, six sequences possess EC cell-binding ability. Specifically, when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold, they mediate cell adhesion. The corresponding soluble peptides block this adhesion, indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR), they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells.

A unique catalytic mechanism for UDP-galactopyranose mutase.

The flavoenzyme uridine 5′-diphosphate (UDP)-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). The latter is an essential precursor to the cell wall arabinogalactan of Mycobacterium tuberculosis. The catalytic mechanism for this enzyme had not been elucidated. Here, we provide evidence for a mechanism in which the flavin cofactor assumes a new role. Specifically, the N5 of the reduced anionic flavin cofactor captures the anomeric position of the galactose residue with release of UDP. Interconversion of the isomers occurs via a flavin-derived iminium ion. To trap this putative intermediate, we treated UGM with radiolabeled UDP-Galp and sodium cyanoborohydride; a radiolabeled flavin-galactose adduct was obtained. Ultraviolet-visible spectroscopy and mass spectrometry indicate that this product is an N5-alkyl flavin. We anticipate that the clarification of the catalytic mechanism for UGM will facilitate the development of anti-mycobacterial agents.

Evolutionary Conservation of Methyl-Accepting Chemotaxis Protein Location in Bacteria and Archaea

The methyl-accepting chemotaxis proteins (MCPs) are concentrated at the cell poles in an evolutionarily diverse panel of bacteria and an archeon. In elongated cells, the MCPs are located both at the poles and at regions along the length of the cells. Together, these results suggest that MCP location is evolutionarily conserved.