Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein

We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K_d) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.     

In vivo glucose monitoring: the clinical reality and the promise

Glucose monitoring is an essential component of modern diabetes management. Three in vivo glucose sensors are now available for clinical use: a subcutaneously implanted amperometric enzyme electrode, a reverse iontophoresis system and a microdialysis-based device. Improvements in glucose-sensing technology continue to be sought, e.g. wired enzyme technology, viscometric affinity sensing and totally implanted glucose sensors. Non-invasive glucose sensing is the ultimate goal of glucose monitoring, but the most investigated approach, near-infrared (NIR) spectroscopy, is presently too imprecise for clinical application. Fluorescence-based glucose sensing offers several advantages and we are investigating strategies which include NIR-based fluorescence resonance energy transfer using concanavalin A/dextran; changes in the intrinsic fluorescence of hexokinase encapsulated in sol-gel; and non-invasive glucose monitoring of cells by measuring glucose-related changes in NADP(H).     

Synthesis and biosensor performance of a near-IR thiol-reactive fluorophore based on benzothiazolium squaraine

Environmentally sensitive near-IR (NIR) dyes are useful fluorophores for various biosensor applications when tissue absorption, scattering, and autofluorescence are a leading concern. Biosensors operating in the NIR region (generally wavelengths >650 nm) would avoid interference from biological media and thereby facilitate relatively interference free sensing. Squaraine dyes are potential candidates to serve as reporter molecules due to their spectral properties in the NIR region, but none is commercially available for site-specific coupling to proteins through native or engineered thiols on cysteine. In this context, we have synthesized a thiol-reactive squaraine that displays fluorescence emission above 650 nm and have coupled the dye site-specifically to various mutants of glucose/galactose binding protein that contained an engineered cysteine for attachment. Mutant E149C/A213R/L238S ISQ GGBP gave a fluorescence change of +50% and a binding constant of 12 mM, which is in the human physiological range for glucose.     

Fluorescence-based glucose sensors

There is an urgent need to develop technology for continuous in vivo glucose monitoring in subjects with diabetes mellitus. Problems with existing devices based on electrochemistry have encouraged alternative approaches to glucose sensing in recent years, and those based on fluorescence intensity and lifetime have special advantages, including sensitivity and the potential for non-invasive measurement when near-infrared light is used. Several receptors have been employed to detect glucose in fluorescence sensors, and these include the lectin concanavalin A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein, and boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor either within a protein which undergoes glucose-induced changes in conformation or because of competitive displacement; measurement of glucose-induced changes in intrinsic fluorescence of enzymes (e.g. due to tryptophan residues in hexokinase) or extrinsic fluorophores (e.g. using environmentally sensitive fluorophores to signal protein conformation). Non-invasive glucose monitoring can be accomplished by measurement of cell autofluorescence due to NAD(P)H, and fluorescent markers of mitochondrial metabolism can signal changes in extracellular glucose concentration. Here we review the principles of operation, context and current status of the various approaches to fluorescence-based glucose sensing.     

Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.     

Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.     

In Vivo image Analysis Using iRFP Transgenic Mice

Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.     

GPT2: a glucose 6-phosphate/phosphate translocator with a novel role in the regulation of sugar signalling during seedling development

Background and Aims

GPT2, a glucose 6-phosphate/phosphate translocator, plays an important role in environmental sensing in mature leaves of Arabidopsis thaliana. Its expression has also been detected in arabidopsis seeds and seedlings. In order to examine the role of this protein early in development, germination and seedling growth were studied.

Methods

Germination, greening and establishment of seedlings were monitored in both wild-type Arabidopsis thaliana and in a gpt2 T-DNA insertion knockout line. Seeds were sown on agar plates in the presence or absence of glucose and abscisic acid. Relative expression of GPT2 in seedlings was measured using quantitative PCR.

Key Results

Plants lacking GPT2 expression were delayed (25–40 %) in seedling establishment, specifically in the process of cotyledon greening (rather than germination). This phenotype could not be rescued by glucose in the growth medium, with greening being hypersensitive to glucose. Germination itself was, however, hyposensitive to glucose in the gpt2 mutant.

Conclusions

The expression of GPT2 modulates seedling development and plays a crucial role in determining the response of seedlings to exogenous sugars during their establishment. This allows us to conclude that endogenous sugar signals function in controlling germination and the transition from heterotrophic to autotrophic growth, and that the partitioning of glucose 6-phosphate, or related metabolites, between the cytosol and the plastid modulates these developmental responses.     

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.     

Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells

Background

Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).

Results

The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.

Conclusion

Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.     

Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor

The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads. The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor. At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent. The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization. As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h. Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column. Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor.     

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

Nanogold-plasmon-resonance-based glucose sensing

Noble metal nanoparticles are well known for their strong interactions with light through the resonant excitations of the collective oscillations of the conduction electrons on the particles, the so-called surface plasmon resonances. The close proximity of two nanoparticles is known to result in a red-shifted resonance wavelength peak, due to near-field coupling. We have subsequently employed this phenomenon and developed a new approach to glucose sensing, which is based on the aggregation and disassociation of 20-nm gold particles and the changes in plasmon absorption induced by the presence of glucose. High-molecular-weight dextran-coated nanoparticles are aggregated with concanavalin A (Con A), which results in a significant shift and broadening of the gold plasmon absorption. The addition of glucose competitively binds to Con A, reducing gold nanoparticle aggregation and therefore the plasmon absorption when monitored at a near-red arbitrary wavelength. We have optimized our plasmonic-type glucose nanosensors with regard to particle stability, pH effects, the dynamic range for glucose sensing, and the observation wavelength to be compatible with clinical glucose requirements and measurements. In addition, by modifying the amount of dextran or Con A used in nanoparticle fabrication, we can to some extent tune the glucose response range, which means that a single sensing platform could potentially be used to monitor microM --> mM glucose levels in many physiological fluids, such as tears, blood, and urine, where the glucose concentrations are significantly different.     

Investigation of the effect of clinically relevant interferents on glucose monitoring using near-infrared spectroscopy

Near infrared spectroscopy (NIR) is a promising technique for continuous blood glucose monitoring for diabetic patients. Four interferents, at physiological concentrations, were introduced to study how the glucose predictions varied with a standard multivariate calibration model. Lactate and ethanol were found to interfere strongly with the glucose predictions unless they were included in the calibration models. Lactate was mistaken for glucose and gave erroneously high glucose predictions, with a dose response of 0.46 mM/mM. The presence of ethanol resulted in too low glucose predictions, with a dose response of −0.43 mM/mM. Acetaminophen, a known interferent in the glucose monitoring devices used for diabetes management today, was not found to be an interferent in NIR spectroscopy, nor was caffeine. Thus, interferents that may appear in high concentrations, such as ethanol and lactate, must be included in the calibration or model building of future NIR-based glucose measurement devices for diabetes monitoring.     

Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose‐responsive polymer‐based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15–31 mM (K_d), which match physiological glucose fluctuations. The dose‐responsive glucose‐mediated release of the novel insulins was demonstrated using glucamine‐derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%–122%) which is unusual for insulin LysB29 derivatives. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Laser tweezers and multiphoton microscopes in life sciences

Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.