Giving cells a new sugar-coating.
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Cell Membrane↗
Biomedical subjects
Mark Howarth
Publications and source records attributed to Mark Howarth.
A monovalent streptavidin with a single femtomolar biotin binding site.
Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.
Animals↗
Targeting quantum dots to surface proteins in living cells with biotin ligase.
Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.
Carbon-Nitrogen Ligases↗
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.
Biotinylation↗
Tapasin enhances MHC class I peptide presentation according to peptide half-life.
Understanding how peptides are selected for presentation by MHC class I is crucial to vaccination strategies based on cytotoxic T lymphocyte priming. We have studied this selection of the MHC class I peptide repertoire in terms of the presentation of a series of individual peptides with a wide range of binding to MHC class I. This series was expressed as minigenes, and the presentation of each peptide variant was determined with the same MHC class I peptide-specific antibody. In wild-type cells, the hierarchy of presentation followed peptide half-life. This hierarchy broke down in cells lacking tapasin but not in cells lacking calreticulin or in cells lacking transporter associated with antigen processing-associated ERp57. We demonstrate a key role for tapasin in shaping the MHC class I peptide repertoire, as enhancement of presentation in the presence of tapasin correlated with peptide half-life.
Amino Acid Sequence↗
Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells.
The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery.
Binding Sites↗
The processing of antigens delivered as DNA vaccines.
The ability of DNA vaccines to provide effective immunological protection against infection and tumors depends on their ability to generate good CD4+ and CD8+ T-cell responses. Priming of these responses is a property of dendritic cells (DCs), and so the efficacy of DNA-encoded vaccines is likely to depend on the way in which the antigens they encode are processed by DCs. This processing could either be via the synthesis of the vaccine-encoded antigen by the DCs themselves or via its uptake by DCs following its synthesis in bystander cells that are unable to prime T cells. These different sources of antigen are likely to engage different antigen-processing pathways, which are the subject of this review. Understanding how to access different processing pathways in DCs may ultimately aid the rational development of plasmid-based vaccines to pathogens and to cancer.
Animals↗
Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin.
MHC class I molecules expressed in a calreticulin-deficient cell line (K42) assembled with beta 2-microglobulin (beta2-m) normally, but their subsequent loading with optimal peptides was defective. Suboptimally loaded class I molecules were released into the secretory pathway. This occurred despite the ability of newly synthesized class I to interact with the transporter associated with antigen processing (TAP) loading complex. The efficiency of peptide loading was reduced by 50%-80%, and impaired T cell recognition was observed for three out of four antigens tested. The peptide-loading function was specific to calreticulin, since the defect in K42 could be rectified by transfection with calreticulin but not a soluble form of calnexin, which shares its lectin-like activity.
Animals↗
DNA transfection screening from single beads.
The solid-phase synthesis of a library of arginine-containing lipid transfection agents on high-loading beads is described. The transfection activity of the cationic lipids was determined using compound cleaved from single beads (single-bead screening) and showed, in some cases, comparable or higher DNA transfection activities as compared to commercially available reagents. Lipids with one arginine headgroup and a cholesterol tail were found to be the most active, even though their DNA binding strength (retardation assays) was relatively weak. Single-bead screening of transfection activity facilitates the rapid analysis of libraries of transfection reagents and will allow the rapid optimization of gene delivery into cells, both in culture and in vivo.
Arginine↗