Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner

2010 ◽  
Vol 431 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Jasmin Strotmeier ◽  
Kwangkook Lee ◽  
Anne K. Völker ◽  
Stefan Mahrhold ◽  
Yinong Zong ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Synaptic Vesicle ◽  
Motor Neurons ◽  
Binding Sites ◽  
Botulinum Neurotoxin ◽  
Specific Binding ◽  
Binding Pocket ◽  
The Other ◽  
Carbohydrate Binding ◽  
Dependent Manner

The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A–G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI–XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).

Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells

1992 ◽  
Vol 9 (2) ◽  
pp. 103-114 ◽  
Author(s):  
C. L. Broadhead ◽  
U. T. O'Sullivan ◽  
C. F. Deacon ◽  
I. W. Henderson
Keyword(s):  
Sodium Chloride ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Binding Sites ◽  
Specific Binding ◽  
Anguilla Anguilla ◽  
Chloride Cells ◽  
Dependent Manner ◽  
Single Class ◽  
Atrial Natriuretic

ABSTRACT The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in freshwater (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.

scholarly journals Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

2006 ◽  
Vol 72 (1) ◽  
pp. 437-442 ◽  
Author(s):  
María A. Ibargutxi ◽  
Anna Estela ◽  
Juan Ferré ◽  
Primitivo Caballero
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Specific Binding ◽  
Significant Inhibition ◽  
The Other ◽  
Cross Resistance ◽  
Earias Insulana ◽  
Cry Proteins ◽  
Cry Toxins ◽  
Laboratory Colony

ABSTRACT Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. 125I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.

Chemoselective cluster labelling of biomolecules

1987 ◽  
Vol 45 ◽  
pp. 760-761
Author(s):  
C. J. Foley ◽  
L. E. Maelia ◽  
J. F. Hainfeld ◽  
J. S. Wall
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Heavy Atom ◽  
The Other ◽  
Biological Structure ◽  
Heavy Atoms ◽  
Specific Binding Sites ◽  
Lower Beam ◽  
High Concentrations ◽  
Beam Dose

The Brookhaven STEM is capable of visualizing single heavy atoms at a beam dose of >103 el/Å2. Heteropolytungstate clusters, including W12PO403, have been found to incorporate several desirable properties as labels for biological specimens. They may be resolved at much lower beam doses due to their high concentrations of multiple heavy atoms and are directly visible labels. A lower beam dose also helps to preserve the biological structure of the specimens. Furthermore, they are extremely stable in the electron beam. Lastly, they are capable of being derivatized as chemoselective reagents for specific binding sites on biomolecules, as in the previously reported undecagold compound.Two new classes of heavy atom labels, one specific for sulfhydryl and the other specific for both amino and sulfhydryl binding sites on proteins, have been synthesized by reactions analogous to those illustrated in Scheme 1.

Contribution of technology to human cell properties

2021 ◽  
pp. 1-4
Author(s):  
Carlota Saldanha
Keyword(s):  
Endothelial Cell ◽  
Human Cell ◽  
Binding Sites ◽  
Blood Cells ◽  
Specific Binding ◽  
Vascular Endothelial Cell ◽  
The Other ◽  
Vascular Endothelial ◽  
Specific Binding Sites ◽  
Human Lungs

After explaining the meaning of SARS-CoV2, the protection rules for the disease caused by this virus are described in order to eradicate the resulting pandemic. Methods to differentiate asymptomatic from symptomatic patients will be mentioned. Human lungs, heart, kidney, endothelium and erythrocyte have specific binding sites for the SARS-CoV2. The aim of this opinion was to highlight some new disposable technology to identify two cell properties. One of them is the vascular endothelial cell (EC) receptor binding to the SARS-CoV2 and the other is related with red blood cells (RBCs) as SARS-CoV2 carrier.

scholarly journals Glycophorin A interferes in the agglutination of human erythrocytes by concanavalin A Explanation of the requirement for enzymic predigestion

1987 ◽  
Vol 241 (2) ◽  
pp. 505-511 ◽  
Author(s):  
S M Gokhale ◽  
N G Mehta
Keyword(s):  
Sialic Acid ◽  
Correlation Coefficient ◽  
Concanavalin A ◽  
Binding Sites ◽  
Band 3 ◽  
Human Erythrocytes ◽  
The Other ◽  
Glycophorin A ◽  
Con A

Human erythrocytes become agglutinable with concanavalin A (Con A) after treatment with various proteinases or neuraminidase. The extent of agglutinability achieved with different enzymes is, however, different: Pronase, papain, trypsin, neuraminidase and chymotrypsin enhance the agglutinability in decreasing order, the last being barely effective. The actions of the enzymes on band 3, the Con A receptor, do not correlate with their abilities to increase the agglutinability: Pronase, papain and chymotrypsin cleave the protein, but not trypsin or neuraminidase. No significant differences are found in the number of Con A-binding sites or the affinities for the lectin between the normal and trypsin- or Pronase-treated cells. Thus the receptor does not seem to play a role in determining the Con A-agglutinability of erythrocytes. On the other hand, the cleavage of glycophorins, especially glycophorin A, and the release of sialic acid (in the peptide-bound form) are well-correlated with the enhancement in agglutination after the action of proteinases. The release of sialic acid by graded neuraminidase digestion and the increase in Con A-agglutinability show a correlation coefficient of 0.88. The major inhibitory role of glycophorin A in the process is indicated by the agglutination of En(a) heterozygous erythrocytes; the cells, known to bear about 50% glycophorin A molecules in their membrane, are agglutinated approximately half as well without proteolysis as are the trypsin-treated cells. Possible mechanisms by which glycophorin A could affect Con A-mediated agglutination are discussed.

Receptors for insulin-like growth factor II in the rat uterus: characterization and variation throughout the estrus cycle

1991 ◽  
Vol 124 (4) ◽  
pp. 434-441 ◽  
Author(s):  
Yallampalli Chandrasekhar ◽  
Satya Narayan ◽  
Pomila Singh ◽  
Manubai Nagamani
Keyword(s):  
Growth Factor ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Cross Linking ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Estrus Cycle ◽  
Rat Uterus ◽  
Factor Ii

Abstract This study was undertaken to identify uterine insulin-like growth factor II receptors and examine the regulation of these receptor levels throughout the estrus cycle. We have demonstrated IGF-II receptors in crude uterine membranes by binding and cross-linking experiments. IGF-II binding to the rat uterine membranes displayed time and temperature dependence and maximum binding was achieved by 2 h at 22°C. Uterine IGF-II binding sites were specific for binding IGF-II peptide and demonstrated negligible binding affinity for IGF-I and no affinity for insulin. The specific anti-IGF-II receptor antibody, R-II-PAB1, blocked the specific [125I]IGF-II binding to uterine membranes in a dose-dependent manner. The characteristics of uterine IGF-II receptor are similar to those reported for other tissues, with a single class of high-affinity binding sites with an apparent dissociation constant of 1.2±0.5 nmol/l and βmax of 2.65±0.41 pmol/mg protein. Affinity cross-linking experiments indicated that the specific binding of [125I]IGF-II in the uterus is associated with a single band of protein with a mol wt of 250 kD. In mature cycling rats, the proestrus uterus had the lowest level of [125I]IGF-II binding per mg membrane protein, without changes in receptor affinity. However, because of greater yield of protein from proestrus uteri, the total [125I]IGF-II binding capacity of the uterus was similar to the other stages of the estrus cycle. These studies demonstrate the presence of authentic IGF-II receptors in the rat uterus and illustrate variations in the concentration of these receptors in the uterus throughout the estrus cycle.

scholarly journals Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland

1998 ◽  
Vol 157 (1) ◽  
pp. 99-106 ◽  
Author(s):  
G Muccioli ◽  
C Ghe ◽  
MC Ghigo ◽  
M Papotti ◽  
E Arvat ◽  
...  
Keyword(s):  
Substance P ◽  
Human Brain ◽  
Pituitary Gland ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Gh Secretagogues ◽  
Specific Binding Sites ◽  
Specific Receptors

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.

Interactions of some partial agonists with high and low affinity binding sites in β-adrenoceptors

1987 ◽  
Vol 65 (1) ◽  
pp. 18-22 ◽  
Author(s):  
I. Takayanagi ◽  
K. Koike ◽  
A. Nakagoshi
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Specific Binding ◽  
The Other ◽  
Affinity Binding ◽  
High Affinity ◽  
Partial Agonists ◽  
Significant Difference ◽  
High Affinity Site ◽  
Derivatives Of

Interactions of derivatives of befunolol (BFE-37, BFE-55, and BFE-61), carteolol, and pindolol with β-adrenoceptors were tested in guinea pig isolated taenia caecum. All the drugs used acted as partial agonists on the β-adrenoceptors when compared with isoprenaline, a full agonist. The pA2 values of BFE-61, carteolol, and pindolol were significantly larger than their pD2 values, while there was no significant difference between the pA2 and pD2 values for BFE-37 and BFE-55. The specific binding of [3H]befunolol to microsomal fractions from the guinea pig taenia caecum distinguished two binding sites, high affinity and low affinity sites. Both sites are considered to be bound by 50 nM of [3H]befunolol. Specific 3H binding was displaced by BFE-61, carteolol, and pindolol in a biphasic manner but in a monophasic manner by BFE-37 and BFE-55. Furthermore, [3H]befunolol binding was only partially displaced by BFE-55 but completely displaced by the other drugs used. These results, together with our previous findings, suggest that BFE-61, carteolol, and pindolol discriminate between the two affinity binding sites in the β-adrenoceptors, which are not discriminated between by BFE-37, and further that BFE-55 may bind with only the high affinity site.

Vitamin E Binds to Specific Binding Sites and Enhances Prostacyclin Production by Cultured Aortic Endothelial Cells

1992 ◽  
Vol 68 (06) ◽  
pp. 744-751 ◽  
Author(s):  
Makoto Kunisaki ◽  
Fumio Umeda ◽  
Toyoshi Inoguchi ◽  
Hajime Nawata
Keyword(s):  
Endothelial Cells ◽  
Vitamin E ◽  
Binding Sites ◽  
Specific Binding ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Specific Binding Sites ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

SummaryWe evaluated the effect of d-α-tocopherol (vitamin E) on the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells. Vitamin E at physiological doses significantly enhanced the production of PGI2 by aortic endothelial cells when added to the culture simultaneously with histamine, the Ca2+ ionophore A23187 (A23187), plasma-derived serum (PDS), or arachidonic acid. This effect was found to occur in a time- and dose-dependent manner, and the maximal enhancement was produced by 9.28 µM of vitamin E for 1 h incubations. Significantly lower amounts of lipid peroxides were measured in endothelial cells stimulated by 10% PDS with 9.28 µM of vitamin E than in those stimulated without vitamin E for over 24 h, although the stimulation during the initial 1 to 12 h period did not have a significant effect on lipid peroxide formation in cultured aortic endothelial cells.We also demonstrated that bovine aortic endothelial cells have specific binding sites for [3H]vitamin E that exhibited time- and temperature-dependent saturability. At 4° C, the nonspecific binding was 8–12% of the total binding, and the specific binding reached equilibrium by 2 h. Specific binding increased with the concentration of [3H]vitamin E and became saturated at concentrations between 1.5 µM and 2.0 µM per 2.0 × 105 cells. Raising the unlabeled vitamin E concentration from 97.7 nM to 1,000 µM reduced the specific binding of 2.0 µM [3H]vitamin E. The Scatchard plot of [3H]vitamin E binding to the endothelial cells shows two classes of binding sites: one with a high affinity {K a1 2.48 ± 0.32 × 107 NT-1, n = 6} and a low capacity {n 1 1.20 ± 0.34 × 107 sites/cell} and the other with a low affinity {K a2 1.18 ± 0.32 × 105 M–1} and a high capacity {n 2 3.39 ± 0.53 × 109 sites/cell}.Our results suggest that the endothelial cells binding sites for vitamin E may play some roles in vascular homeostasis in vivo, and that vitamin E may prevent the development of atherosclerotic changes due in part to the enhancement of PGI2 production by the vascular wall and its action as an antioxidant in vascular endothelial cell.

scholarly journals Normal cellular prion protein is a ligand of selectins: binding requires LeX but is inhibited by sLeX

2007 ◽  
Vol 406 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chaoyang Li ◽  
Poki Wong ◽  
Tao Pan ◽  
Fan Xiao ◽  
Shaoman Yin ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Fusion Protein ◽  
Human Brain ◽  
Prion Protein ◽  
Fusion Proteins ◽  
Cellular Prion Protein ◽  
The Other ◽  
Dependent Manner ◽  
Peripheral Tissues ◽  
Sialyl Lex

The normal PrPC (cellular prion protein) contains sLeX [sialyl-LeX (Lewis X)] and LeX. sLeX is a ligand of selectins. To examine whether PrPC is a ligand of selectins, we generated three human PrPC–Ig fusion proteins: one with LeX, one with sLeX, and the other with neither LeX nor sLeX. Only LeX-PrPC–Ig binds E-, L- and P-selectins. Binding is Ca2+-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLeX-PrPC–Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrPC. The selectins precipitated PrPC in human brain in a Ca2+-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrPC precipitated. Therefore the presence of sialic acid prevents the binding of PrPC in human brain to selectins. Hence, human brain PrPC interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

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