Biotin Derivatives Research Articles

Surface Expression of Epithelial Na Channel Protein in Rat Kidney

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

Zinc(II)-Coordinated Oligotyrosine: A New Class of Cell Penetrating Peptide

A new series of cell penetrating peptides (CPPs) are described. The peptides are oligomers of Tyr-ZnDPA, a tyrosine derivative with an appended 2,2'-dipicolylamine unit that forms a very stable coordination complex with a zinc (II) cation. This in turn allows reversible association with a chelating oxyanion such as a carboxylate or phosphate derivative. The peptide oligomers (Tyr-ZnDPA) n where n = 1, 2, 4, 8, are highly water soluble, but upon association with fatty acids or phospholipids they partition into an organic octanol phase. Furthermore, a fluorescent, fluorescein-labeled version of the octamer, (Tyr-ZnDPA) 8-Fl, can enter living mammalian cells via endocytosis and a biotin derivative can deliver fluorescein-labeled streptavidin. Fluorescence microscopy and flow cytometry experiments show that cell uptake is diminished by conditions that inhibit endocytosis. Additionally, uptake of (Tyr-ZnDPA) 8-Fl is greater than fluorescein labeled octaarginine (Arg 8-Fl) in all cell lines tested (CHO, COS-7, HeLa). Another difference with Arg 8-Fl is that cell uptake of (Tyr-ZnDPA) 8-Fl does not require the presence of heparan sulfate proteoglycans on the cell surface. This difference may eventually be of practical value because drug delivery systems that employ alternative endocytic mechanisms may be optimal for different cell lines or they may deliver selectively to different organelles within a cell.

Nanodiamond bioconjugate probes and their collection by electrophoresis

The application of detonation nanodiamonds (NDs) as probes for protein capture and electrophoretic collection was investigated. NDs were chemically modified in a series of reactions to produce a ND-NH 2 product that had increased chemical homogeneity. The product was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). FTIR spectra were taken using an IR vacuum cuvette and the samples were dehydrated at different temperatures. The ND-NH 2 product was capable of conjugating to N-hydroxysuccinimide derivatives of TAMRA and biotin. We calculated that the number of chemically attached TAMRA molecules on ND-NH 2 was ∼ 1 molecule/nm 2. The singly conjugated TAMRA-ND (T-ND) and doubly conjugated TAMRA-ND-Biotin (T-ND-B) products formed stable aqueous colloidal suspensions. T-ND and T-ND-B were collected on planar electrodes and silicon field tip arrays using a field of 10 V/cm. The rate of collection for the aminated ND is dependent upon field strength and an exponential decrease in current was observed as a function of time. Streptavidin was captured by the T-ND-B bioconjugate probe and this nanoparticle–protein complex was collected from solution by electrophoresis.

Expanding the Substrate Tolerance of Biotin Ligase through Exploration of Enzymes from Diverse Species

Technologies that enable the site-specific conjugation of chemical probes onto proteins are extremely useful for applications in cell biology and proteomics. We cloned, expressed, and purified biotin ligases from nine different species and screened them for the ability to ligate unnatural analogues of biotin onto the human p67 biotin acceptor domain. We discovered that the biotin ligases of Saccharomyces cerevisiae (yeast) and Pyrococcus horikoshii could accept alkyne and azide derivatives of biotin, respectively. HPLC, gel-shift, and mass spectrometry assays confirmed that these ligation reactions were ATP- and enzyme-dependent, as well as site-specific. We used the P. horikoshii-catalyzed azide ligation reaction to site-specifically introduce a phosphine probe onto p67 using the Staudinger ligation. These new ligation reactions demonstrate the differential substrate specificities of biotin ligases from different organisms and open the door to novel protein labeling applications.

Salt-induced Changes in the Plasma Membrane Proteome of the Halotolerant Alga Dunaliella salina as Revealed by Blue Native Gel Electrophoresis and Nano-LC-MS/MS Analysis

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.

Biotinylated Anisomycin: A Comparison of Classical and “Click” Chemistry Approaches

Two approaches to the synthesis of biotinylated derivatives of the stress-activated protein kinase (SAPK) pathway activator anisomycin have been investigated. Attachment of the biotin moiety to the central core was achieved either through the use of a classical displacement reaction on alpha-halo carbonyl derivatives of biotin or through a copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition ("click") coupling of biotinylated azides to propargyl-marked analogues of anisomycin. In each case, the resultant N-linked molecular probes were found to be active in SAPK pathway immunoblot assays, while their O-linked counterparts were inactive. However, in sharp contrast to the classical coupling approach which results in low coupling yields, the aqueous "click" coupling process was found to deliver high yields of biotinylated probes, making it the conjugation method of choice. A survey of the available methods for the addition of a propargyl marker onto a range of chemical functionalities strongly suggests that this copper(I)-catalyzed 1,3-dipolar Huisgen cycloaddition approach to biotinylation may be generally applied.

Trafficking of a Secretory Granule Membrane Protein Is Sensitive to Copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.

Use of Biotin Derivatives to Probe Conformational Changes in Proteins

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys(826) in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83-87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Site-directed biotinylation of antibodies for controlled immobilization on solid surfaces

Site-directed biotinylation of antibodies at the hinge region was developed to immobilize antibodies in an oriented manner via biotin–streptavidin linkage. When intact antibody was biotinylated with maleimide-activated biotin after reduction, the reaction preferentially occurred at the sulfhydryl groups between the C H1 and the C L domains and, provided that the reagent concentration exceeded a certain level, at those between the C H2 and the C H2 domains at the hinge. Based on this result, we devised an approach in which free maleimide was added to compete with the activated biotin for the preferential sites between the C H1 and the C L domains. Since the smaller molecular size of free maleimide made it more accessible for the reaction than biotin, maleimide bound to the groups between the C H1 and the C L domains first and thus conceded the groups between the C H2 and the C H2 domains to biotin under optimal conditions. In an alternative approach, selective biotinylation at the hinge was also achieved by reacting activated biotin with F(ab′) 2 fragment prepared by enzymatic cleavage. This result indicated that, when free of Fc, the hinge structure, which contains the functional groups, of the fragment was open, allowing easy access to the biotin derivative from the aqueous medium. Both site-directed biotinylation preparations were tested as capture antibodies in sandwich-type immunoassays and compared to whole antibody randomly biotinylated at amino groups on the molecule. Preparations of both the intact antibody and the F(ab′) 2 showed consistently enhanced detection capabilities that were 2.6 and 20 times that of the control, respectively.

Functional Interrogation of the Kinome Using Nucleotide Acyl Phosphates

The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin–quencher is bound to Eu–streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher–biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2–2 nmol L −1. In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay.

A new tool for the rational design of methylbiotin hosts

To study the binding mode of biotin related compounds with artificial hosts, we have developed a new tool to be used as a guide to test their behaviour prior to their synthesis. In that way, we have considered a set of 23 complexes comprising biotin and urea derivatives with synthetic hosts to develop a Partial Least Squares Cross-Validated (PLS-CV) model. The data, for such a model, are the binding constants ( K b) of each complex and the interaction energies (− E min) calculated by molecular mechanics with AMBER and OPLS force fields. The predictive power of the model has been verified.

Biotin Reagents for Antibody Pretargeting. 7. Investigation of Chemically Inert Biotinidase Blocking Functionalities for Synthetic Utility

An investigation was conducted to evaluate three biotin derivatives designed to block biotinidase cleavage of the biotinamide bond. Difficulties in multistep syntheses of molecules containing tert-butyl protected hydroxymethyl and carboxylate groups positioned alpha to a biotinamide bond led to the investigation of alternative biotinidase-blocking moieties that do not require protection and deprotection. The targeted biotin derivatives contained serine-O-methyl ether, 2-aminobutyric acid, and valine moieties conjugated to the biotin carboxylate functionality. Those derivatives were further modified with a radioiodinated aryl ring to study their biotinidase stability. As a comparison to previously studied biotin derivatives, radioiodinated versions of biotin conjugates that contained (a) no biotinidase stabilizing group, (b) an N-methyl (sarcosine) stabilizing group, (c) an alpha-carboxylate (aspartate) stabilizing group and hydroxymethyl (serine) stabilizing group were also prepared and tested. When tested in human serum, all of the radioiodinated biotinidase-stabilized biotin derivatives had <1% biotinamide cleavage. Thus, under the conditions studied, all of the tested biotinidase blocking moieties appeared to be equal with regards to protection from biotinidase cleavage. Further testing of the biotin derivatives included a HPLC assay to determine their relative dissociation from recombinant streptavidin (rSAv). The dissociation of cyanocobalamin (CN-Cbl) adducts of biotin-serine-O-methyl ether, biotin-aminobutyric acid, and biotin-valine were compared with the CN-Cbl adduct of biotin-sarcosine. The relative rates of dissociation found were biotin-sarcosine-CN-Cbl > biotin-valine-CN-Cbl > biotin-serine-O-methyl ether-CN-Cbl > biotin-aminobutyric acid-CN-Cbl. Due to the high cost of serine-O-ethyl ether (and its N-Boc derivative) and difficulty in syntheses of its biotin derivatives, that adduct is not an attractive candidate for application to compounds used in vivo. The higher lipophilicity and diminished binding of the biotin-valine adduct also makes its use in vivo less attractive. Thus, the biotin-aminobutyric acid adduct appears to be the best candidate for incorporation into biotin derivatives used in vivo, as it simplifies the synthetic procedures, has low cost, and provides effective blocking of biotinidase while retaining high binding affinity.

Thiol-Mediated Anchoring of Ligands to Self-Assembled Monolayers for Studies of Biospecific Interactions

We report a method to immobilize thiol-containing ligands onto self-assembled monolayers (SAMs) of alkanethiolates presenting chloracetylated hexa(ethylene glycol) groups. The chloroacetyl groups react with thiols under mild basic conditions, enabling the stable immobilization of biologically active ligands in a well-defined orientation. These SAMs on gold are well suited for studies of biospecific interactions of immobilized ligands with proteins and cells. As a demonstration, we functionalized these SAMs with thiol-containing derivatives of biotin and benzene sulfonamide and observed the specific binding of neutravidin and carbonic anhydrase, respectively. We also used this method to generate mixed SAMs presenting the Arg-Gly-Asp (RGD) peptide sequence and demonstrated the integrin-mediated adhesion of fibroblast cells to these SAMs. This approach would allow the immobilization of proteins and other sensitive biomolecules and ligands for a wide variety of applications in biotechnology.

CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Synthesis and characterization of thermo‐ and pH‐sensitive block copolymers bearing a biotin group at the poly(ethylene oxide) chain end

AbstractA poly(ethylene oxide)‐block‐poly(dimethylamino ethyl methacrylate) block copolymer (PEO‐b‐PDMAEMA) bearing an amino moiety at the PEO chain end was synthesized by a one‐pot sequential oxyanionic polymerization of ethylene oxide (EO) and dimethylamino ethyl methacrylate (DMAEMA), followed by a coupling reaction between its PEO amino and a biotin derivative. The polymers were charac terized with 1H NMR spectroscopy and gel permeation chromatography. Activated biotin, biotin‐NHS (N‐hydroxysuccinimide), was used to synthesize biotin‐PEO‐PDMAEMA. In aqueous media, the solubility of the copolymer was temperature‐ and pH‐sensitive. The particle size of the micelle formed from functionalized block copolymers was determined by dynamic light scattering. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3552–3558, 2006

A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.

Syntheses of Functionalized Biotin N-1‘ Derivatives: New Tools for the Control of Gene Expression with Small Molecules

The exceptionally high affinity of biotin toward avidin and streptavidin is at the basis of (strept)avidin-biotin biotechnology, which has numerous applications in life sciences. Recent biotin developments for in vivo and in vitro acylation of selective targeted protein and intein-mediated site specific protein biotinylation require the free biotin carboxyl function to covalently bind with the targeted protein. However, recently this carboxylic function has been used to substitute biotin with numerous ligands and flags. In the present work, we propose the N-1' labeling possibilities of biotin, keeping the valeric chain free. We describe liquid and solid-phase syntheses of functionalized biotin N-1' derivatives. Although the N-1' modification involves a two-log decrease in affinity, in vitro these molecules kept their high avidin affinity (around 10(-12) M) and the in vivo acylation ability of new biotin derivatives.

Lipoplexes with biotinylated transferrin accessories: Novel, targeted, serum-tolerant gene carriers

Novel transfecting assemblies comprising biotinylated cationic liposomes, DNA and tribiotinylated transferrin-streptavidin (streptavidin(bio 3-transferrin)) accessories have been prepared, characterized and evaluated for toxicity and DNA delivery capability in human cervical carcinoma cells (HeLa). Two new lipophilic cholesteryl-based biotin derivatives, biotinylcholesterylformylhydrazide (MSB1) and aminohexanoylbiotinylcholesterylformylhydrazide (MSB2) provided docking points for streptavidin(bio 3-transferrin) on cationic liposomes which were formulated with N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09) and dioleoylphosphatidylethanolamine (DOPE) in a 2:48:50 molar ratio. Ethidium dye displacement assays and gel retardation studies suggest that in ternary complexes, the DNA is electrostatically bound to the cationic liposomes while transferrins remain liposome-bound through streptavidin–biotin interactions. Assemblies fully protected plasmid DNA from serum nuclease digestion over a range of liposome:pGL3 DNA ratios (3–8:1, w/w) and exhibited low growth inhibition of HeLa cells (circa 5%) at the optimal transfection composition for streptavidin(bio 3-transferrin):liposome:pGL3 DNA of 10:6:1 (w/w/w). Transfection levels, which were twice those of untargeted lipoplexes containing MSB1 or MSB2, were not significantly diminished in the presence of 10% foetal bovine serum. Excess transferrin (200 μg per well) reduced transfection levels to those of untargeted complexes, supporting the notion that at least 50% of ternary complexes gained entry into the cervical carcinoma cells by receptor mediation. Conversely, transfection levels with untargeted lipoplexes were only slightly reduced in the presence of transferrin at the same concentration.

GM-CSF-surface-modified B16.F10 melanoma cell vaccine

Through the specific and tight interaction between streptavidin and biotin, a novel platform was developed to allow for rapid (less than 2 h), efficient and durable display of streptavidin-tagged bioactive GM-CSF on the surface of biotinylated B16.F10 tumor cells. This technology involved biotinylation of the cell membrane with a biotin derivative and surface modification of the biotinylated cells with the bi-functional fusion protein, streptavidin-tagged GM-CSF. Furthermore, the resultant GM-CSF-modified B16.F10 whole tumor cell vaccine could induce strong and long-lasting systemic protection against the wild-type tumor challenge. Therefore, the platform may represent a fast, efficient and safe approach for the whole tumor cell vaccination.