Catabolism of desialylated human plasma alpha1-antitrypsin and its trypsin complex in the rat.

Human plasma α₁-antitrypsin (α₁-AT) was labeled with either ³H [³H-labeled NANA (N-acetyl-neuraminic acid)-7] residues in the carbohydrate moiety) or ¹⁴C (?-N-methyl-[¹⁴C]lysyl residues in the protein backbone) or with both isotopes in the corresponding residues. After intravenous injection into rats of the doubly labeled partially (50%) desialylated (methyl-[¹⁴C]·[³H]NANA-7)-α₁-AT, the rates of disappearance from the plasma of both isotopes were very rapid and yielded essentially the same circulatory half-life of 5 min. The rapid disappearance of the doubly labeled glycoprotein from the plasma was accompanied by concomitant fast and equal accumulations of ¹⁴C and ³H in the liver which constituted about 70% of the administered dose 15 min after the injection. The asialo (methyl-[¹⁴C])-α₁-AT·trypsin complex or methyl-[¹⁴C]-α₁-AT·trypsin complex had a plasma survival time (45 min) that was intermediate between methyl-[¹⁴C]-α₁-AT and its desialylated derivative. These complexes were removed from the plasma by the liver (45% of the injected dose 60 min after injection), although not as rapidly as asialo (methyl-[¹⁴C])-α₁-AT. Blockade of the reticuloendothelial (Kupffer) cells by simultaneous injection of heat-denatured albumin inhibited the liver uptake of the inhibitor·trypsin complexes but not that of the uncomplexed asialo α₁-AT. Radioactive ?-N,N-dimethyllysine, ?-N-monomethyllysine, methionine, choline, and betaine were separated and identified from the trichloro-acetic acid-soluble fraction of rat livers 25 min after injection of asialo (methyl-[¹⁴C])-α₁-AT.     

The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure:

with Fuc present on only about 50% of the oligosaccharides.     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Reaction of turbulence at the edge and in the center of the plasma column to pulsed impurity injection caused by the sputtering of the wall coating in L-2M stellarator

Impurity injection into plasma caused by the sputtering of the wall coating in the L-2M stellarator during auxiliary electron cyclotron resonance heating leads to a change in the level of plasma density fluctuations with frequencies above 0.25 MHz: suppression of long-wavelength (k _⊥ = 2 cm^–1) density fluctuations in the edge plasma, intensification of short-wavelength (k _⊥ = 30 cm^–1) and long-wavelength (k _⊥ = 1 cm^–1) fluctuations at the midradius of the plasma column, and intensification of short-wavelength fluctuations (k _⊥ = 20 cm^–1) in the plasma center (including the gyroresonance region). At the same time, the level of fluctuations with frequencies below 0.25 MHz remains unchanged. In the edge plasma, a decrease in the plasma potential and suppression of its fluctuations is observed during impurity injection, which also causes an increase in MHD activity.     

Osmotic water permeability in glycoprotein containing liposomes

The kinetics of osmotic water permeability in proteoliposomes containing ₁-acid glycoprotein was investigated by means of stopped-flow spectrophotometry. A biphasic time-course of scattered light with time was registered. The rate constants calculated from fits to an exponential function in the first phase were proportional to the final medium osmolarity. The apparent second order rate constants K_app (Osm^-1 sec^-1) were determined at different glycoprotein concentrations in the original mixture for preparation of proteoliposomes. The value of K_app at lipid:glycoprotein weight ratio = 1 was plotted in Arrhenius coordinates. The calculated activation energy for water permeation through the lipid bilayer suggests that eventual channel mechanism may be involved due to the presence of glycoprotein molecule in the liposomes.     

The role of <Emphasis Type="Italic">N</Emphasis>-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

Background

Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.

Results

We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN⁺ cells and transmission of DC-SIGN-captured virions to CD4⁺ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic III_B and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, III_B and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1_ADA contained gp120 levels similar to the gp120 levels in WT HIV-1_ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.

Conclusions

We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

     

Sigma-1 receptor deficiency reduces MPTP-induced parkinsonism and death of dopaminergic neurons

Sigma-1 receptor (σ₁R) has been reported to be decreased in nigrostriatal motor system of Parkinson''s disease patients. Using heterozygous and homozygous σ₁R knockout (σ₁R^+/− and σ₁R^−/−) mice, we investigated the influence of σ₁R deficiency on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-impaired nigrostriatal motor system. The injection of MPTP for 5 weeks in wild-type mice (MPTP-WT mice), but not in σ₁R^+/− or σ₁R^−/− mice (MPTP-σ₁R^+/− or MPTP-σ₁R^−/− mice), caused motor deficits and ~40% death of dopaminergic neurons in substantia nigra pars compacta with an elevation of N-methyl-d-aspartate receptor (NMDAr) NR2B phosphorylation. The σ₁R antagonist NE100 or the NR2B inhibitor Ro25-6981 could alleviate the motor deficits and the death of dopaminergic neurons in MPTP-WT mice. By contrast, MPTP-σ₁R^+/− mice treated with the σ₁R agonist PRE084 or MPTP-σ₁R^−/− mice treated with the NMDAr agonist NMDA appeared to have similar motor deficits and loss of dopaminergic neurons as MPTP-WT mice. The pharmacological or genetic inactivation of σ₁R suppressed the expression of dopamine transporter (DAT) in substantia nigra, which was corrected by NMDA. The activation of σ₁R by PRE084 enhanced the DAT expression in WT mice or σ₁R^+/− mice. By contrast, the level of vesicular monoamine transporter 2 (VMAT2) in σ₁R^+/− mice or σ₁R^−/− mice had no difference from WT mice. Interestingly, MPTP-WT mice showed the reduction in the levels of DAT and VMAT2, but MPTP-σ₁R^−/− mice did not. The inactivation of σ₁R by NE100 could prevent the reduction of VMAT2 in MPTP-WT mice. In addition, the activation of microglia cells in substantia nigra was equally enhanced in MPTP-WT mice and MPTP-σ₁R^−/− mice. The number of activated astrocytes in MPTP-σ₁R^−/− mice was less than that in MPTP-WT mice. The findings indicate that the σ₁R deficiency through suppressing NMDAr function and DAT expression can reduce MPTP-induced death of dopaminergic neurons and parkinsonism.Parkinson''s disease (PD) is a neurodegenerative disorder characterized by motor symptoms, including bradykinesia and tremor, and a progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc).^{1, 2} Sigma-1 receptor (σ₁R), previously named the opioid receptor sigma-1, is found primarily in motoneurons localized in the brainstem and spinal cord.³ The σ₁R is expressed in dopaminergic neurons and astrocytes.⁴ The σ₁R agonist PRE084 has been reported to exert neurorestorative effects on 6-hydroxydopamine (6-OHDA)-induced parkinsonism.⁴ Using positron emission tomography, the σ₁R-binding sites are found to be reduced in the brains of early-phase PD patients.⁵ However, the influence of σ₁R deficiency on the pathogenesis of PD has not yet been reported.Dopamine toxicity is involved in the etiology of PD.⁶ The σ₁R-binding sites on dopaminergic nerve terminals are involved in increasing dopamine release by enhancing N-methyl-d-aspartate receptors (NMDAr).⁷ The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolized to 1-methyl-4-phenylpyridinium in glial cells selectively impairs dopaminergic neurons in SNpc through disrupting respiratory enzymes and causing oxidative damage.⁸ The dopamine transporter (DAT), a high-affinity transmembrane protein, is responsible for dopamine reuptake from the synaptic cleft and the transportation of 1-methyl-4-phenylpyridinium into dopaminergic nerve terminals.⁹ The σ₁R is co-expressed with DAT in dopaminergic neurons.⁴ Furthermore, the low density of DAT has been confirmed in the brains of PD patients.⁵The activation of σ₁R enhances the Ca²⁺ influx across NMDAr through increasing the phosphorylation of NR2B or the trafficking NMDAr to the plasma membrane.^{10, 11} The NMDAr NR2B inhibitor can attenuate MPTP- or 6-OHDA-induced parkinsonian symptoms and neurodegeneration.¹² The σ₁R deficiency has been demonstrated to reduce Aβ-induced neuronal cell death through suppressing NR2B phosphorylation.¹³ The inflammation is a predominant aspect of PD, manifested by glial activation with the expression of pro-inflammatory mediators.¹⁴ Sustained neuro-inflammation can exacerbate the degeneration of dopaminergic neurons.¹⁵ The blockade of σ₁R has been reported to inhibit methamphetamine-induced astrogliosis.¹⁶ Moreover, the 6-OHDA-induced spontaneous rotations or decline of dopaminergic fibers in σ₁R knockout mice seem to be less than those in wild-type (WT) mice.⁴ Paquette et al. reported that the blockade of σ₁R could attenuate abnormal involuntary movements induced by 6-OHDA.¹⁷In this study, we employed heterozygous and homozygous σ₁R knockout (σ₁R^+/− and σ₁R^−/−) mice to investigate the influence of σ₁R deficiency on MPTP-induced parkinsonism and death of dopaminergic neurons, and the underlying molecular mechanisms. Using the experimental PD models of MPTP-treated σ₁R^+/− mice and σ₁R^−/− mice, the present study provides in vivo evidence that the σ₁R deficiency through suppressing NMDAr function and DAT expression can attenuate MPTP-induced dopaminergic neurodegeneration and parkinsonism.     

Physical Parameters of a Reactor-Stellarator with Small Ripples of the Helical Magnetic Field

The paper describes the calculation data on the physical parameters of a reactor-stellarator, where the nonuniformities of the helical field are smaller than the toroidal magnetic field nonuniformities: ε_h < ε_t. Unlike the previous studies, where the ion-component transport coefficients had the collision frequency dependence proportional to ν^1/2, this being equivalent to the ε_h > ε_t case, in the present calculations, these coefficients were assumed to be in proportion to the first power of the collision frequency, D_i ∝ ν for ν_eff < 2ω_E, and to D_i ∝ ν^?1 for the inverse inequality. Here, ω_E is the rotation frequency of plasma in the radial electric field. As before, the plasma electrons corresponded to the mode of D_e ∝ ν^?1. As initial parameters for numerical calculations, a reactor with R = 8 m, r_p = 2 m, and B₀ = 5 Т was taken. A numerical code was used to solve the set of equations that describes the plasma space?time behavior in the reactor-stellarator under the conditions of equal diffusion fluxes. The start of reactor operation in the mode of thermonuclear burning was provided by heating sources with a power of several tens of megawatts. Steady-state operating conditions of a self-sustained thermonuclear reaction were attained by maintaining the plasma density through DT fuel pellet injection into the plasma.     

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

Background

Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed.

Methods

The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins.

Results and conclusions

Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA.

General significance

This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.     

Surface glycoproteins of resting and activated human T lymphocytes

Summary This review summarizes some recent studies on the surface glycoproteins of human thymocytes and T lymphocytes. Purified cells were surface labeled by the galactose oxidase-NaB³H₄ or periodate-NaB³H₄ techniques. The radioactive membrane glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Thymocytes and T lymphocytes show characteristic surface glycoprotein profiles which are easily distinguishable from those of the other main groups of human leukocytes. We observed specific changes in the surface glycoprotein patterns which correlate with the degree of maturation and functional activation of T cells. Surface molecules carrying T cell specific antigens were identified by immune-precipitation from lysates of surface labeled thymocytes and T lymphocytes using rabbit anti-human T cell antibodies. Finally we describe a leukocyte membrane glycoprotein which is a precursor of serum ₁ acid glycoprotein (orosomucoid).     

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[1-¹⁴C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between G_M1 and G_D1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse₅Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse₅Cer backbone. No cross-reaction with G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Circular dichroism of human urinary Tamm-Horsfall glycoprotein

Summary Human urinary Tamm-Horsfall glycoprotein, which contains 28% carbohydrate, has a monomeric molecular weight of about 80,000 but is isolated from urine in the form of intertwining helical suprastructures with molecular weights greater than 10⁷. The native glycoprotein was dissociated and denatured with 6 M guanidinium chloride and was subsequently renatured by dialysis against a Tris-HCl buffer. Using sedimentation equilibrium, the renatured glycoprotein was characterized by a of 256,800 and a of 356,000. The ratio,M _z/M _w, of 1.39 indicates some polydispersity with regard to molecular size. There was no evidence of helical suprastructures in the renatured glycoprotein as judged by electron microscopy. Ca²⁺ concentrations of up to 50 mM failed to precipitate the renatured glycoprotein; in contrast, the native glycoprotein is precipitated by Ca²⁺ concentrations between 5–10 mM. The circular dichroic spectrum of renatured Tamm-Horsfall glycoprotein was obtained, resolved, and tentative band assignments made. The spectrum, which is quite similar to that of native Tamm-Horsfall glycoprotein, exhibited negative extrema at 269 nm (due in large part to disulfides and tyrosines) and at 215 nm (due to protein-structure and the N-acetylated hexosamines). The-helical content of the glycoprotein was estimated to be no more than 10% and the amount of-structure to be about 33%; these values were not affected by the presence of Ca²⁺ (1 mM). A glycopeptide fraction (ca. 90% carbohydrate), prepared by extensive pronase digestion of the reduced, S-carboxymethylated glycoprotein, exhibited an ellipticity extremum at 212 nm of +4,750 deg · cm²/dmole, referred to the concentration of (N-acetylated) hexosamines and neuraminic acid.Research Career Development Awardee (AM-00055).     

Rate of synthesis of βL-lipovitellin in the liver of immature chicks treated with 17β estradiol

Using a combination of radioimmunoprecipitation and SDS-polyacrylamide gel electrophoresis of the immunoprecipitate we studied the rate of synthesis of the heavy chain of β-lipovitellin in the liver of immature chicks. In male and female chicks the base-line synthesis of β_L-lipovitellin¹ was about 30 molecules per minute and per cell. Four days after a single injection of 40 mg estradiol/kg, as many as 48,000 molecules of β_L-lipovitellin were synthesized per minute and per diploid liver cell. The increase in the rate of β_L-lipovitellin synthesis could be correlated with an increase in membrane bound mRNA coding for β_L-lipovitellin.     

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION*

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca²⁺-ATPase E1PCa₂·Mg, a complex of E1Ca₂·BeF_x, was successfully developed by addition of beryllium fluoride and Mg²⁺ to the Ca²⁺-bound state, E1Ca₂. In E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, two Ca²⁺ are occluded at high affinity transport sites, its formation required Mg²⁺ binding at the catalytic site, and ADP decomposed it to E1Ca₂, as in E1PCa₂·Mg. Organization of cytoplasmic domains in E1Ca₂·BeF_x was revealed to be intermediate between those in E1Ca₂·AlF₄⁻ ADP (transition state of E1PCa₂ formation) and E2·BeF₃⁻·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca₂·BeF_x in contrast to no superfluorescence of TNP-AMP bound to E1Ca₂·AlF_x. E1Ca₂·BeF_x with bound TNP-AMP slowly decayed to E1Ca₂, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF₃⁻, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca₂·BeF_x mimics E1PCa₂·Mg, and between those of E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻. E1Ca₂·BeF_x at low 50–100 μm Ca²⁺ was converted slowly to E2·BeF₃⁻ releasing Ca²⁺, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. Ca²⁺ replacement of Mg²⁺ at the catalytic site at approximately millimolar high Ca²⁺ decomposed E1Ca₂·BeF_x to E1Ca₂. Notably, E1Ca₂·BeF_x was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca²⁺ with 15 mm Mg²⁺. Also, stable E1Ca₂·BeF_x was produced from E2·BeF₃⁻ at 0.7 mm lumenal Ca²⁺ by binding two Ca²⁺ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca²⁺ → E1PCa₂·Mg.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of the P-type ion transporting ATPases, catalyze Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). The enzyme forms phosphorylated intermediates from ATP or P_i in the presence of Mg²⁺ (10–13). In the transport cycle, the enzyme is first activated by cooperative binding of two Ca²⁺ ions at high affinity transport sites (E2 to E1Ca₂, steps 1–2) (14) and autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon this E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). Subsequent isomeric transition to the ADP-insensitive form (E2PCa₂), i.e. loss of ADP sensitivity at the catalytic site, results in rearrangement of the Ca²⁺ binding sites to deocclude Ca²⁺, reduce the affinity, and open the lumenal gate, thus releasing Ca²⁺ into the lumen (E2P, steps 4–5). Finally Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form the Ca²⁺-unbound inactive E2 state (steps 6 and 7). Mg²⁺ bound at the catalytic site is required as a physiological catalytic cofactor in phosphorylation and dephosphorylation and thus for the transport cycle. The cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and absence of Ca²⁺, and subsequent Ca²⁺ binding at lumenally oriented low affinity transport sites of E2P reverses the Ca²⁺-releasing step and produces E1PCa₂, which is then decomposed to E1Ca₂ by ADP.Open in a separate windowFIGURE 1.Ca²⁺ transport cycle of Ca²⁺-ATPase.Various intermediate structural states in the transport cycle were fixed as their structural analogs produced by appropriate ligands such as AMP-PCP (non-hydrolyzable ATP analog) or metal fluoride compounds (phosphate analogs), and their crystal structures were solved at the atomic level (15–22). The three cytoplasmic domains, N, P, and A, largely move and change their organization state during the transport cycle, and the changes are coupled with changes in the transport sites. Most remarkably, in the change from E1Ca₂·AlF₄⁻·ADP (the transition state for E1PCa₂ formation, E1PCa₂·ADP·Mg^‡) to E2·BeF₃⁻ (the ground state E2P·Mg) (23–25), the A domain largely rotates by more than 90° approximately parallel to the membrane plane and associates with the P domain, thereby destroying the Ca²⁺ binding sites, and opening the lumenal gate, thus releasing Ca²⁺ into the lumen (see Fig. 2). E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP without Mg²⁺ is nearly the same as E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP in their crystal structures (17, 18, 22).Open in a separate windowFIGURE 2.Structure of SERCA1a and its change during processing of phosphorylated intermediate. E1Ca₂·AlF₄⁻·ADP (the transition state analog for phosphorylation E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (the ground state E2P analog (25)) were obtained from the Protein Data Bank (PDB accession code 1T5T (17) and 2ZBE (21), respectively). Cytoplasmic domains N (nucleotide binding), P (phosphorylation), and A (actuator), and 10 transmembrane helices (M1–M10) are indicated. The arrows on the domains, M1′ and M2 (Tyr¹²²) in E1Ca₂·AlF₄⁻·ADP, indicate their approximate motions predicted for E1PCa₂·ADP·Mg^‡ → E2P·Mg. The phosphorylation site Asp³⁵¹, TGES¹⁸⁴ of the A domain, Arg¹⁹⁸ (tryptic T2 site) on the Val²⁰⁰ loop (DPR¹⁹⁸AV²⁰⁰NQD) of the A domain, and Thr²⁴² (proteinase K site) on the A/M3-linker are shown. Seven hydrophobic residues gather in the E2P state to form the Tyr¹²²-hydrophobic cluster (Y122-HC); Tyr¹²²/Leu¹¹⁹ on the top part of M2, Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² of the A domain, and Val⁷⁰⁵/Val⁷²⁶ of the P domain. The overall structure of E1Ca₂·AlF₄⁻·ADP is virtually the same as those of E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN (17, 18, 22).Despite these atomic structures, yet unsolved is the structure of E1PCa₂·Mg, the genuine physiological intermediate E1PCa₂ with bound Mg²⁺ at the catalytic site without the nucleotide. Its stable structural analog has yet to be developed. E1PCa₂·Mg is the major intermediate accumulating almost exclusively at steady state under physiological conditions. Its rate-limiting isomerization results in Ca²⁺ deocclusion/release producing E2P·Mg as a key event for Ca²⁺ transport. In E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP, and E1PCa₂·Ca·AMP-PN, the N and P domains are cross-linked and strongly stabilized by the bound nucleotide and/or Ca²⁺ at the catalytic site, thus they are crystallized (17, 18, 22). Kinetically, E1PCa₂·Ca formed with CaATP is markedly stabilized due to Ca²⁺ binding at the catalytic Mg²⁺ site, and its isomerization to E2P is strongly retarded in contrast to E1PCa₂·Mg (26, 27). Thus, the bound Ca²⁺ at the catalytic Mg²⁺ site likely produces a significantly different structural state from that with bound Mg²⁺.Therefore, it is now essential to develop a genuine E1PCa₂·Mg analog without bound nucleotide and thereby gain further insight into the structural mechanism in the Ca²⁺ transport process. It is also crucial to further clarify the structural importance of Mg²⁺ as the physiological catalytic cation. In this study, we successfully developed the complex E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, as the E1PCa₂·Mg analog by adding beryllium fluoride (BeF_x) to the E1Ca₂ state without any nucleotides. For its formation, Mg²⁺ binding at the catalytic site was required and Ca²⁺ substitution for Mg²⁺ was absolutely unfavorable, revealing a likely structural reason for its preference as the physiological cofactor. In E1Ca₂·BeF₃⁻, two Ca²⁺ ions bound at the high affinity transport sites are occluded. It was also produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, mimicking E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. All properties of the newly developed E1Ca₂·BeF₃⁻ fulfilled the requirements as the E1PCa₂·Mg analog, and hence we were able to uncover the hitherto unknown nature of E1PCa₂·Mg as well as structural events occurring in the phosphorylation and isomerization processes. Also, we successfully found the conditions that perfectly stabilize the E1Ca₂·BeF₃⁻ complex.     

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca²⁺]_i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca²⁺]_i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca²⁺ into CMT1A SC. Correction of the altered [Ca²⁺]_i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca²⁺]_i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.Charcot-Marie-Tooth disease type 1 (CMT1)³ is a progressive hereditary motor and sensory neuropathy, characterized by distal muscle wasting and weakness, foot deformities, and severe slowing of nerve conduction, because of progressive demyelination (1). With a prevalence of 1 case in 2500, CMT1 is the most common hereditary neurologic disorder, and in the majority of cases (CMT1A) the disease is associated with a duplication on chromosome 17p11.2 of the gene for PMP22 (peripheral myelin protein 22) (2). PMP22 is a 22-kDa glycoprotein mainly expressed by myelinating Schwann cells (SC) and localized in compact myelin (3). The transgenic rat model of CMT1A, obtained by overexpression of PMP22 (4), confirms a role of PMP22 in the pathogenesis of CMT1A. Both PMP22 overexpression because of gene duplication and point mutations of PMP22 are associated with a CMT1A phenotype.The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear. Some reports indicate that a perturbed homeostasis of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) might be causally involved in the demyelination process. Conditions inducing an increased [Ca²⁺]_i in SC impair cell differentiation and myelination (5, 6), similarly to what occurs in CMT1A. Incubation of intact rat nerves with Ca²⁺ and ionophores causes a progressive demyelination, spreading from the paranodes and invading regions of formerly compact myelin, which is dependent upon a rise in the [Ca²⁺]_i of SC (5).Additional evidence for the detrimental effect of a [Ca²⁺]_i elevation on myelin production by SC comes from application of ATP to murine SC monocultures, inducing an immediate and large increase in the [Ca²⁺]_i. As a result of ATP treatment, maturation and differentiation of SC, as well as expression of the myelin basic protein and production of compact myelin, are completely prevented (6). Taken together, the above observations indicate that abnormally elevated Ca²⁺ levels are causally related to impairment of myelin production by SC.In this study, we addressed the possible correlation between PMP22 overexpression and alteration of the [Ca²⁺]_i homeostasis in SC from a rat model of CMT1A. We recorded higher levels of basal [Ca²⁺]_i in affected than in control cells, and we identified the mechanisms responsible for the perturbation of the [Ca²⁺]_i levels in CMT1A SC. Experiments with pharmacological inhibitors and with small interfering RNA (siRNA) unequivocally demonstrated a correlation in CMT1A SC between overexpression of the purinergic receptor P2X7 and influx of extracellular [Ca²⁺]_i across this plasma membrane receptor/channel. In addition, correction of the abnormally elevated [Ca²⁺]_i levels by the use of a P2X7 antagonist or through down-regulation of the expression of P2X7 by transfection with siRNA or with short hairpin RNA-expressing plasmid (shRNA) restored the normal phenotype in CMT1A SC. These findings suggest that CMT1A should be considered as a “calcium disease.” Identification of P2X7 activation as the pathogenetic mechanism underlying demyelination may provide the rationale for a new therapeutic strategy for CMT1A, a disease with no currently available treatment.     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.