BBA - General Subjects (v.1770, #9)
Editorial Board (ii).
Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5 by D.A. Greer; S. Ivey (1275-1282).
The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans — one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing β(1–4)-N-acetyl-d-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galβ(1–4)–GlcNAcx component found in other Pgps.
Keywords: P-glycoprotein; Multidrug resistance; MES-SA/Dx5 cell line; DIG glycan detection; Lectin; Gel elution;
Identification of palmitate-regulated genes in HepG2 cells by applying microarray analysis by Christina Vock; Mareike Gleissner; Maja Klapper; Frank Döring (1283-1288).
Palmitate is the most abundant saturated fatty acid in the human diet and the major one synthesized de novo. To identify palmitate-regulated genes we performed whole genome mRNA expression profiling by using human hepatoma HepG2 cells. We identified eleven genes which are significantly (single-sided permutational t-test, p < 0.05) regulated by low concentration of palmitate (50 μM). We observed a decreased expression of five metallothioneins, and an increased expression of liver expressed plasminogen activator inhibitor-1 protein and insulin-like growth factor II, which play a prominent role in the development of the metabolic syndrome. Comparative promoter analysis in-silico revealed common transcriptional regulation of differentially expressed genes through erythroid kruppel-like factor and members of the zinc binding protein factor family. In conclusion, low physiological palmitate concentrations changed expression of very responsive genes.
Keywords: Gene expression; Hepatocytes; Microarray; Palmitate; Transcription factor;
Induction of histone acetylation on the CRBPII gene in perinatal rat small intestine by Yuko Ogura; Kazuki Mochizuki; Toshinao Goda (1289-1296).
The expression of genes associated with lipid and vitamin A metabolism is elevated when the small intestinal mucosa is maturing rapidly during the perinatal period. We have previously reported that cellular retinol-binding protein type II (CRBPII) mRNA levels rise abruptly in the rat small intestine during this period. In this study, we examined whether the acetylation of histones H3 and H4 is involved in the intestinal expression of CRBPII during the perinatal stage. The expression of cyclin D1 and cyclin B1 genes, which are markers of cell proliferation, decreased markedly during the perinatal period, whereas expression of CRBPII as well as villin, a marker of intestinal maturation, increased rapidly. Using a ChIP assay, we showed rapid induction of acetylation of the histones H3 and H4 which interacted with the promoter/enhancer region of the CRBPII gene at this time. The binding of CBP and p300, which have histone acetyltransferase activity, as well as binding of retinoid X receptor α (RXRα) increased on the CRBPII promoter/enhancer region during the perinatal period. These results suggest that CRBPII gene expression during the perinatal period is associated with abrupt acetylation of histones H3 and H4 followed by the binding of CBP/p300 and RXRα.
Keywords: Histone acetylation; CRBPII; CBP/p300; Intestine; Development;
Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum by Krishna Samanta; Pulak Kar; Biswarup Ghosh; Tapati Chakraborti; Sajal Chakraborti (1297-1307).
Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na2CO3 and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.
Keywords: m-calpain; Calpastatin; Calpain–calpastatin association; Endoplasmic reticulum; Pulmonary artery; Smooth muscle;
Neuroprotective effect of individual ginsenosides on astrocytes primary culture by M. Victoria Naval López; M. Pilar Gómez-Serranillos Cuadrado; Olga M. Palomino Ruiz-Poveda; Angel M. Villar Del Fresno; M. Emilia Carretero Accame (1308-1316).
Most of the known pharmacological effects of Panax ginseng on the central nervous system are due to its major components – ginsenosides. Although the antioxidant ability of ginseng root has already been established, this activity has never been evaluated for isolated ginsenosides on astrocytes. The activity of protopanaxadiols Rb1, Rb2, Rc and Rd, and protopanaxatriols Re and Rg1 was evaluated in vitro on astrocytes primary culture by means of an oxidative stress model with H2O2. The viability of astrocytes was determined by the MTT reduction assay and by the LDH release into the incubation medium. The effects on the antioxidant enzymes catalase, superoxide dismutase (SOD), glutathione peroxidases (GPx) and glutathione reductase (GR) and on the intracellular reactive oxygen species (ROS) formation were also investigated. Exposure of astrocytes to H2O2 decreased cell viability as well as the antioxidant enzymes activity and increased ROS formation. Oxidative stress produced significant cell death that was reduced by previous treatment with the tested ginsenosides. Ginsenosides Rb1, Rb2, Re and Rg1 were effective in reducing astrocytic death, while Rb1, Rb2, Rd, Re and Rg1 decreased ROS formation, ginsenoside Re being the most active. Ginsenosides from P. ginseng induce neuroprotection mainly through activation of antioxidant enzymes.
Keywords: Panax ginseng C.A. Meyer; Ginsenoside; Astrocyte; Neuroprotection;
Physical characterization of the stratum corneum of an in vitro psoriatic skin model by ATR-FTIR and Raman spectroscopies by Geneviève Bernard; Michèle Auger; Jacques Soucy; Roxane Pouliot (1317-1323).
The stratum corneum is an important permeability barrier for the skin. The disorganization of the skin protective barrier characterizes some skin diseases such as psoriasis. Indeed, psoriatic skin is known to be more permeable than normal human skin. An in vitro human skin substitute may be obtained by the auto-assembly method. This method was adapted to produce psoriatic substitutes. FTIR spectroscopy is a well-established method to evaluate the order of hydrocarbon chains in terms of population of trans and gauche conformers. Using ATR-FTIR, we have compared the physicochemical properties of the stratum corneum in skin models derived from uninvolved and involved psoriatic cells with those derived from normal cells. Our results suggest that the stratum corneum of involved psoriatic skin substitutes is less organized than that of normal skin substitutes. Also, it seems that the properties of uninvolved psoriatic skin may vary with seriousness of the disease. The development of a new psoriatic skin model would be helpful in the design of new treatments and to increase the understanding of the mechanisms of this pathology.
Keywords: Tissue Engineering; Psoriasis; Stratum corneum; Lipids; FTIR spectroscopy; Raman spectroscopy;
Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell by B. Griffaut; E. Debiton; J.C. Madelmont; J.C. Maurizis; G. Ledoigt (1324-1330).
Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW < 10 kDa), such as salicylic acid, can enhance this activity.
Keywords: Helianthus tuberosus; Plant stress; Superoxide dismutase; Alkaline phosphatase; Tumour;
(LYS)16-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells by Alan L. Parker; Lorna Eckley; Surjeet Singh; Jon A. Preece; Louise Collins; John W. Fabre (1331-1337).
Extracellular stability, endocytic escape, intracellular DNA release and nuclear translocation of DNA are all critical properties of non-viral vector/DNA particles. We have evaluated a (Lys)16-based linear, reducible polycation (RPC) in combination with an acid-dependent, anionic fusogenic peptide for gene delivery to dividing and post-mitotic cells. The RPC was formed from Cys(Lys)16Cys monomers. Molecular weight was 24,000 Da, corresponding to an average of 10.5 peptide monomers per RPC. Non-reducible polylysine (PLL) (27,000 Da) and monomeric (Lys)16 peptide were evaluated for comparison. (Lys)16/DNA particles were disrupted at fusogenic peptide concentrations well below those used for gene delivery. By contrast, RPC/DNA an PLL/DNA particles were stable in the presence of high concentrations of the anionic peptide. Addition of 10% serum virtually abolished the transfection ability of (Lys)16/DNA/fusogenic peptide particles, but had little effect on RPC/DNA/fusogenic peptide particles. RPC/DNA/fusogenic peptide particles were highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium. PLL/DNA/fusogenic peptide particles were moderately effective on cell lines, but gave no gene delivery with corneal endothelial cells. We conclude that (Lys)16-based RPC/DNA/fusogenic peptide particles provide a gene delivery system which is potentially stable in the extracellular environment and, on reductive depolymerisation, can release DNA plasmids for nuclear translocation.
Keywords: Reducible polycation; Fusogenic peptide; Post-mitotic cell; Lysine; Nuclear translocation; Non-viral gene delivery;
Biomineralization: Functions of calmodulin-like protein in the shell formation of pearl oyster by Zhenguang Yan; Zi Fang; Zhuojun Ma; Jinye Deng; Shuo Li; Liping Xie; Rongqing Zhang (1338-1344).
Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.
Keywords: Calmodulin-like protein; Biomineralization; Organic layer; Pearl oyster; Pinctada fucata;
Human NPC1L1 and NPC1 can functionally substitute for the ncr genes to promote reproductive development in C. elegans by Marsha M. Smith; Diane J. Levitan (1345-1351).
The NPC1 and NPC1L1 are related genes whose general role is in cholesterol trafficking. However, reduction of activity of these genes results in very different phenotypes. Niemann–Pick C disease type 1 is a neurodegenerative disease with no current treatment, where cholesterol accumulates in lysosomes. The disease arises due to autosomal recessive mutations in the NPC1 gene. The NPC1L1 gene has recently been identified as the target for the drug ezetimibe (Zetia), a cholesterol absorption inhibitor, and has been shown to be an intestinal cholesterol transporter. We demonstrate that human NPC1L1, as well as human NPC1, can functionally substitute for the Caenorhabditis elegans genes ncr-1 and/or ncr-2. These genes are known to play a role in the process of dauer formation, a process which can be modulated by cholesterol in sensitized genetic backgrounds. Our results demonstrate that these human proteins retain some functional conservation, though their biological roles are vastly different.
Keywords: NPC1L1; NPC1; NCR genes; Ezetimibe; Dauer;
Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line by Elizandra Braganhol; Alessandra S.K. Tamajusuku; Andressa Bernardi; Márcia R. Wink; Ana M.O. Battastini (1352-1359).
Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5′-NT/CD73. Quercetin was able to inhibit the ecto-5′-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (α,β-methyleneadenosine-5′-diphosphate), an ecto-5′-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5′-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.
Keywords: Glioma; Quercetin; Ecto-5′-NT/CD73; Adenosine; Cell proliferation;
Investigations of combinations of mutations in the jellyfish green fluorescent protein (GFP) that afford brighter fluorescence, and use of a version (VisGreen) in plant, bacterial, and animal cells by Prapapan Teerawanichpan; Travis Hoffman; Paula Ashe; Raju Datla; Gopalan Selvaraj (1360-1368).
Among the GFPs used for imaging green fluorescence, the Emerald version has been considered the best GFP to use but there is no formal report on its construction or the relevance of the amino acid (aa) substitutions in it relative to the commonly used GFPs. Here, we have shown that a version of Emerald makes Escherichia coli host cells visibly green even under dim room light conditions. Exploiting this feature, we have determined for the first time whether the changes in the structure of Emerald protein brought about by the aa substitutions are all indeed essential for brightness. F64L and S72A accompanying the classical S65T substitution on the chromophore-bearing helix are essential. Two amino acid changes, one on the surface (N149K) of the β barrel that encases the helix and the other (I167T) near the chromophore enhance the visible green colour individually and additively when present together. The other two substitutions, M153T (on the surface) and H231L (on the surface), do not contribute to the visible green phenotype, even though in earlier studies M153T has been reported to enhance GFP fluorescence. The GFP version with F64L–S65T–S72A–N149K–I167T is referred to as VisGreen. We found VisGreen and Emerald to be indistinguishable in their quantum yield, molar extinction coefficient, folding efficiency, or photosensitivity. VisGreen rendered bacterial, plant, and animal cells highly fluorescent. Interestingly, N149K in the above combination was not essential to render bacterial cells highly fluorescent.
Keywords: Bright green fluorescence; Imaging; Animals; Plants; Bacteria; GFP; Combinatorial amino acid substitutions; Emerald; VisGreen;
Inhibition of vascular endothelial growth factor receptor tyrosine kinases impairs adipose tissue development in mouse models of obesity by H.R. Lijnen; B. Van Hoef; D. Kemp; D. Collen (1369-1373).
We have studied the effect of PTK787 (Vatalanib), an inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, on adipose tissue development. Oral administration of PTK787 for 4 weeks (2 mg/g high fat diet, HFD) to C57Bl/6 mice resulted in a significant reduction in total body weight and of subcutaneous (SC) and gonadal (GON) adipose tissue mass, as compared to control animals fed HFD only (all p < 0.0005). In the GON adipose tissue adipocytes were hypertrophic after PTK787 treatment. Blood vessel size and density were not significantly affected by PTK787 treatment. Expression of Flk-1 (VEGFR-2) mRNA was significantly reduced in SC and GON adipose tissues of PTK787 treated mice. De novo fat pad formation following injection of preadipocytes in NUDE mice was significantly (p < 0.005) impaired by PTK787 administration (2 mg/g HFD for 4 weeks), without associated effect on blood vessel size or density. Thus, in nutritionally induced murine obesity models, oral administration of the VEGFR tyrosine kinases inhibitor PTK787 resulted in reduced adipose tissue development.
Keywords: Vascular endothelial growth factor (VEGF); VEGF receptor; Adipose tissue; Obesity; Tyrosine kinase;
Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications by Ylva Ivarsson; Malena A. Norrgård; Ulf Hellman; Bengt Mannervik (1374-1381).
Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.
Keywords: Enantioselectivity; Epoxide resolution; Glutathione transferase; Protein modification; Rational redesign;
Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species by Subash Chandra Gupta; Hifzur Rahman Siddique; Neeraj Mathur; Achchhe Lal Vishwakarma; Ranjit Kishore Mishra; Daya Krishna Saxena; Debapratim Kar Chowdhuri (1382-1394).
We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg 9 to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.
Keywords: Dichlorvos; Hsp70; Oxidative stress; ROS; Apoptosis; Transgenic Drosophila;
Involvement of Ca2+/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus by Isamu Kameshita; Yusuke Yamada; Tetsuyuki Nishida; Yasunori Sugiyama; Noriyuki Sueyoshi; Akira Watanabe; Yasuhiko Asada (1395-1403).
Although multifunctional Ca2+/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl2, suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca2+/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl2 in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.
Keywords: Basidiomycete; Ca2+/calmodulin-dependent protein kinase; Coprinus cinereus; Mushroom; Protein phosphorylation;
Purification and characterization of lectin from fruiting body of Ganoderma lucidum by Atul Thakur; Monika Rana; T.N. Lakhanpal; Absar Ahmad; M.I. Khan (1404-1412).
A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.
Keywords: Mushroom; Ganoderma lucidum; Purification; Thermodynamic properties; Glycan binding;
Metal binding ability of glutathione transferases conserved between two animal species, the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum by Masafumi Yoshinaga; Tatsuya Ueki; Hitoshi Michibata (1413-1418).
Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II) > V(IV) > Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.
Keywords: Glutathione transferase (GST); Vanabin; Metal selectivity; Immobilized metal ion affinity chromatography (IMAC); Ascidian; Schistosome;
Specific binding and self-structure induction to poly(A) by the cytotoxic plant alkaloid sanguinarine by Prabal Giri; Gopinatha Suresh Kumar (1419-1426).
The cytotoxic plant alkaloid sanguinarine was found to bind preferentially and strongly to single stranded poly(A) with an association constant (Ka) in the range 3.6–4.6 × 106 M− 1 in comparison to several nucleic acids. The binding induced unique self-structure formation in poly(A) that showed cooperative melting transition in circular dichroism, absorbance, and differential scanning calorimetry studies. The alkaloid binding was characterized to be intercalation as revealed from fluorescence quenching experiments and was predominantly enthalpy driven as revealed from isothermal titration calorimetry. Sanguinarine is the first and only natural product so far known to induce a self-structure formation in poly(A).
Keywords: Sanguinarine; poly(A); Specific binding; Self-structure formation; Spectroscopy; Thermodynamics;
Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis by Małgorzata Przybyło; Danuta Martuszewska; Ewa Pocheć; Dorota Hoja-Łukowicz; Anna Lityńska (1427-1435).
The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.
Keywords: β1–6 branching; Glycosylation; Integrins; Melanoma; MS/MS analysis; PHA-L;