European journal of biochemistry最新文献

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

The neutral N-glycan structures of Arion lusitanicus (gastropod) skin, viscera and egg glycoproteins were examined after proteolytic digestion, release of the glycans from the peptides, fluorescent labelling with 2-aminopyridine and fractionation by charge, size and hydrophobicity to obtain pure glycan structures. The positions and linkages of the sugars in the glycan were analysed by two dimensional HPLC (size and hydrophobicity) and MALDI-TOF mass spectrometry before and after digestion with specific exoglycosidases. The most striking feature in the adult tissues was the high amount of oligomannosidic and small paucimannosidic glycans terminated with 3-O-methylated mannoses. The truncated structures often contained modifications of the inner core by beta1,2-linked xylose to the beta-mannose residue and/or an alpha-fucosylation (mainly alpha1,6-) of the innermost GlcNAc residue. Skin and viscera showed predominantly the same glycans, however, in different amounts. Traces of large structures carrying 3-O-methylated galactoses were also detected. The egg glycans contained mainly (approximately 75%) oligomannosidic structures and some paucimannosidic structures modified by xylose or alpha1,6-fucose, but in this case no methylation of any monosaccharide was detected. Thus, gastropods seem to be capable of producing many types of structures ranging from those typical in human to structures similar to those found in nematodes, and therefore will be a valuable model to understand the regulation of glycosylation. Furthermore, this opens the way for using this organism as a host for the production of recombinant proteins. The detailed knowledge on glycosylation also may help to identify targets for pest control.

In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD(50) values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin - a urease isoform from jackbean seeds that displays a toxic effect in mice (LD(50) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x mL(-1) for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni(+2) and Zn(+2) ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.

SUT2 was found in a screen for multicopy suppressors of the synthetic slow growth phenotype of a Deltaras2Deltagpa2 double deletion mutant. It failed, however, to cure the lethal phenotype of a Deltaras1Deltaras2 mutant suggesting that it acts upstream of Ras or in a parallel pathway. By testing cAMP-dependent reactions including the accumulation of storage carbohydrates, pseudohyphal differentiation, entry of meiosis as well as the measurement of FLO11 reporter activity we show that Sut2p modulates the activity of protein kinase A (PKA). Additionally, we demonstrate that cellular levels of Ras2p are affected by Sut2p and that Sut2-GFPp accumulates significantly in the nucleus. Based on the observed influence of high SUT2 gene dosage on PKA activity as well as Sut2p's homology to the presumptive transcription factor Sut1p, we suggest that Sut2p contributes to regulation of PKA activity at the level of transcription.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.

The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.

The chemical structure of the free lipid A isolated from Mesorhizobium huakuii IFO 15243(T) was elucidated. Lipid A is a mixture of at least six species of molecules whose structures differ both in the phosphorylation of sugar backbone and in fatty acylation. The backbone consists of a beta (1'-->6) linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide that is partly substituted by phosphate at position 4'. The aglycon of the DAG-disaccharide has been identified as alpha-D-galacturonic acid. All lipid A species carry four amide-linked 3-hydroxyl fatty residues. Two of them have short hydrocarbon chains (i.e. 3-OH-i-13:0) while the other two have longer ones (i.e. 3-OH-20:0). Distribution of 3-hydroxyl fatty acids between the reducing and nonreducing DAG is symmetrical. The nonpolar as well as (omega-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties. Two acyloxyacyl residues occur exclusively in the nonreducing moiety of the sugar backbone but their distribution has not been established yet. The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted by ester-linked acyl components.

Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.