BBA - General Subjects (v.1726, #1)

Selenocysteine in proteins—properties and biotechnological use by Linda Johansson; Guro Gafvelin; Elias S.J. Arnér (1-13).
Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK a of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.
Keywords: Selenocysteine; Selenoprotein; Selenium biochemistry; Biotechnology; Enzymology; Synthetic and recombinant selenoprotein production;

Retinol induces permeability transition and cytochrome c release from rat liver mitochondria by Fábio Klamt; Marcos Roberto de Oliveira; José Cláudio Fonseca Moreira (14-20).
Biological actions of retinoids on modulation of cellular gene expression by nuclear receptors are widely known. Recently, extra-nuclear effects of retinoids have been proposed, but remain to be better elucidated. Considering that retinoids induce apoptosis in tumor cells by an unknown mechanism, and that mitochondria play a key role in controlling apoptosis via cytochrome c (cyt c) release, we exposed rat liver mitochondria to 3–40 μM of retinol (vitamin A), and observed that retinol causes mitochondrial permeability transition (MPT) and cyt c release, in a concentration-dependent pattern. Increased superoxide anion generation and lipoperoxidation were also observed. Cyclosporin A or trolox co-administration reverted all parameters tested. In view of these findings, we conclude that retinol induces mitochondria oxidative damage, leading to MPT and cyt c release by opening of the permeability transition pore, thus suggesting a putative mechanism of apoptosis activation by retinol.
Keywords: Vitamin A; Retinol; Oxidative stress; Mitochondria; Cytochrome c; Permeability transition pore; MPT;

Screening for and purification of novel self-aggregatable lectins reveal a new functional lectin group in the bark of leguminous trees by Chieko Ina; Kotone Sano; Makiko Yamamoto-Takahashi; Hiroko Matsushita-Oikawa; Hiroko Takekawa; Yayoi Takehara; Haruko Ueda; Haruko Ogawa (21-27).
A solubility–insolubility transition assay was used to screen the bark and stems of seven leguminous trees and plants for self-aggregatable lectins. Novel lectins were found in two trees, Robinia pseudoacacia and Wisteria floribunda, but not in the leguminous plants. The Robinia lectin was isolated from coexisting lectin by combined affinity chromatographies on various sugar adsorbents. The purified lectins proved to be differently glycosylated glycoproteins. One lectin exhibited the remarkable characteristics of self-aggregatable lectins: localization in the bark of legume trees, self-aggregation dissociated by N-acetylglucosamine/mannose, and coexistence with N-acetylgalactosamine/galactose-specific lectins, which are potential endogenous receptors. Self-aggregatable lectins are a functional lectin group that can link enhanced photosynthesis to dissociation of glycoproteins.
Keywords: Lectin; Self-aggregation; Endogenous receptor; Legume bark; Robinia pseudoacacia; Wisteria floribunda;

Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells by Toshiyuki Fukuda; Kazuhiro Takekoshi; Toru Nanmoku; Kiyoaki Ishii; Kazumasa Isobe; Yasushi Kawakami (28-33).
The small GTPase, RhoA, and its downstream effecter Rho-kinase (ROK) are reported to be involved in various cellular functions, such as myosin light chain phosphorylation during smooth muscle contraction and exocytosis. Indeed, growing evidence suggests that the RhoA/Rho-kinase pathway plays an important role in regulating exocytosis in these cells. However, it is not known whether the RhoA/Rho-kinase pathway has an effect on catecholamine synthesis. Using the rat pheochromocytoma cell line, PC12, we examined the effects of either Rho-kinase inhibitor (Y27632) or RhoA inhibitor (C3 toxin) on nicotine-induced catecholamine biosynthesis. We show that nicotine (10 μM) induces a significant, though transient, increase in RhoA activation in these cells. Treatment with either Y27632 (1 μM) or C3 toxin (10 μg/ml) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH) mRNA and the corresponding enzyme activity. TH catalyzes the rate-limiting step in the biosynthesis of catecholamine. Y27632 significantly inhibited nicotine-induced phosphorylation of TH at Ser40 as well as Ser19, which are known to be phosphorylated by Ca2+/calmodulin kinase II. Furthermore, Y27632 (10 μM) as well as C3 toxin (10 μg/ml) significantly inhibited the nicotine-induced increase of TH at the protein level. Thus, we propose that activation of RhoA, and its downstream effecter Rho-kinase, is a prerequisite for catecholamine biosynthesis in PC12 cells. At the concentrations used in our experiments, Y27632 does not affect cAMP/PKA activity or PKC activity, indicating that the inhibitory effect of Y27632 can be attributed to the inhibition of Rho-kinase activity as observed in chromaffin cells. In contrast, neither Y27632 (10 μM) nor C3 toxin (10 μg/ml) significantly altered catecholamine secretion in PC12 cells. In conclusion, we have demonstrated that inhibition of the Rho/Rho-kinase pathway in chromaffin cells lowers TH activity, probably through CaMKII inhibition. By contrast, neither Y27632 nor C3 toxin affect the secretion of catecholamine.
Keywords: RhoA/Rho kinase system; Tyrosine hydroxylase (TH); PC12 cell; Y27632; C3 toxin;

Discovery of novel inhibitor of human leukocyte common antigen-related phosphatase by Xiaoning Yang; Jingya Li; Yueyang Zhou; Qiang Shen; Jiwu Chen; Jia Li (34-41).
Human leukocyte common antigen-related phosphatase (LAR) may play a role in type 2 diabetes and cancer, and in the development of the nervous system, and it may be an attractive target for the treatment of diabetes and cancer. We identified eight hits from the random screening of LAR D1 with a high-throughput screening assay. Further validation of the eight hits showed that the meD insertion was associated with inhibition of LAR D1D2 and LAR D1Q. These data suggest that the inserted meD peptide influences the interaction of the enzyme and inhibitor, which is consistent with the kinetic catalysis constants of the substrate pNPP. Our data showed that Hit 1, the first published novel inhibitor of LAR, is a competitive inhibitor with a K i of 330 nM that displays obvious selectivity for LAR and mouse PTPσ, but not for other protein tyrosine phosphatases.
Keywords: LAR; Protein tyrosine phosphatase; High-throughput screening; Competitive; Inhibitor; meD peptide;

Background: We have previously shown that there were great discrepancies between different agents regarding their glutathione stimulating potential and that agents with mainly oxidative effects did not increase concentrations of glutathione in human cell cultures, in contrast to other thiol reactive agents. In order to evaluate whether increased glutathione degradation might be one reason for these discrepancies, we have investigated the effect of different agents with potential influence on glutathione metabolism in human cell cultures with or without acivicin inhibition of γ-glutamyltranspeptidase (GT), since GT is responsible for the initial degradation of glutathione. Methods: Intra- and extracellular concentrations of glutathione were investigated in HeLa and hepatoma cell cultures, with and without acivicin inhibition of GT, in the presence of oxidative and electrophilic agents (copper ions, hydrogen peroxide and N-ethylmaleimide), hydroquinone, reducing agents (lipoic acid and N-acetylcysteine), and a thiol reactive metal (mercury ions). Results: There were great discrepancies between the different agents regarding their maximal glutathione response (the sum of the intracellular and the extracellular amount of glutathione) in cell cultures. There was only a small increase in total glutathione in the presence of hydrogen peroxide or N-ethylmaleimide before the cell protein decreased compared to findings with mercury ions, lipoic acid or hydroquinone. In both HeLa and hepatoma cell cultures, there were correlations between the original glutathione amount and the total glutathione amount observed after acivicin inhibition. Conclusion: The relatively small increase of glutathione amount in the presence of oxidative and electrophilic agents compared to other thiol reactive agents is not due to increased GT degradation of glutathione.
Keywords: Electrophilic agent; γ-glutamyltranspeptidase; Glutathione; Human cell line; Mercury ion; Oxidative agent;

Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells by Jorge R. Toledo; Oliberto Sánchez; Raquel Montesino Seguí; Yaiza Fernández García; María P. Rodríguez; José A. Cremata (48-56).
The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.
Keywords: Adenovirus; Transduction; Milk; Erythropoietin; Glycosylation; Mammary gland;

Preparation and characterization of active site protected poly(ethylene glycol)–avidin bioconjugates by Stefano Salmaso; Alessandra Semenzato; Sara Bersania; Marco Chinol; Giovanni Paganelli; Paolo Caliceti (57-66).
Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin–PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin–PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin–PEG 10 and 20 kDa prepared in the presence of imminobiotin–PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79–85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin–PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.
Keywords: Avidin; Poly(ethylene glycol); Bioconjugation; PEGylation; Active site protection; Avidin/biotin interaction;

Purification and characterization of a novel lipase from the digestive glands of a primitive animal: The scorpion by Nacim Zouari; Nabil Miled; Slim Cherif; Hafedh Mejdoub; Youssef Gargouri (67-74).
Higher animal's lipases are well characterized, however, much less is known about lipases from primitive ones. We choose the scorpion, one of the most ancient invertebrates, as a model of a primitive animal. A lipolytic activity was located in the scorpion digestive glands, from which a scorpion digestive lipase (SDL) was purified. Pure SDL, a glycosylated protein, has a molecular mass of 50 kDa, it presents the interfacial activation phenomenon. It was found to be more active on short-chain triacylglycerols than on long-chain triacylglycerols. SDL is a serine enzyme and possesses one accessible sulfhydryl group which is not essential for the catalysis. Among the NH2-terminal 33 residues, a 17 amino acids sequence shows similarities with sequence of Drosophila melanogaster putative lipase. Interestingly, neither colipase, nor bile salts were detected in the scorpion hepatopancreas. This indicates that colipase evolved in vertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. Furthermore, polyclonal antibodies directed against SDL failed to recognise the classical digestive lipases. Altogether, these results suggest that SDL is a member of a new group of digestive lipases belonging to invertebrates.
Keywords: Scorpio maurus; Scorpion; Hepatopancreas; Digestive lipase; Purification; Characterization;

Biochemical and enzymatic characterization of two basic Asp49 phospholipase A2 isoforms from Lachesis muta muta (Surucucu) venom by Daniela C.S. Damico; Sérgio Lilla; Gilberto de Nucci; Luis A. Ponce-Soto; Flávia V. Winck; José Camillo Novello; Sérgio Marangoni (75-86).
Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I) → Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P  < 0.05) the enzymatic activity of LmTX-I, suggesting that the binding site for crotapotin in this PLA2 was similar to another in the basic PLA2 of the crotoxin complex from C. durissus cascavella venom.
Keywords: Characterization; Enzyme; Lachesis muta muta; Phospholipase A2; Snake venom;

Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties by Samuel Dufour; Magali Deleu; Katherine Nott; Bernard Wathelet; Philippe Thonart; Michel Paquot (87-95).
New linear analogs of surfactin have been synthesized. Their physico-chemical parameters were determined. The results indicate that these linear products show surface activities although they are lowered compared to those of cyclic compounds. The hemolytic activities have also been assayed. In contrast with cyclic surfactins, no significant hemolysis occurs for the linear products in the range of concentrations tested. Moreover, a protective effect against Triton X-100 induced hemolysis has been highlighted for linear surfactins. The concentration at which this protective effect happens is correlated directly to the CMC, and inversely to the acyl chain length of the product. In a hypotonic medium, analogs having a long acyl chain tend to increase the hemolysis, meanwhile the product with the shortest chain tends to decrease it.
Keywords: Surfactin; CMC; Hemolysis; Surfactin analog;

Acrosome reaction in bovine spermatozoa: Role of reactive oxygen species and lactate dehydrogenase C4 by C. O'Flaherty; E. Breininger; N. Beorlegui; M.T. Beconi (96-101).
After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process.
Keywords: Oxidoreductase; Oxidative process; Reducing equivalent; Reactive oxygen species; Protein kinase; Bovine spermatozoa;

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix–loop–helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the α-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic α-helical peptides generated from a helix–loop–helix domain (residues 1–38 of cLZ) by nicking at leucine17. A third peptide consisting of an α-helix (residues 18–38) and a two-stranded β-sheet (residues 39–56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.
Keywords: Lysozyme; Muramidase; Proteolysis; Pepsin; Antimicrobial activity; Peptide motif; Membrane damage; Human breast milk; Gastrointestinal mucosa;

Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate by Kyung-Hee Park; Yong-Doo Park; Jae-Rin Lee; Hwa-Sun Hahn; Sang-Jin Lee; Chang-Dae Bae; Jun-Mo Yang; Dong-Eun Kim; Myong-Joon Hahn (115-120).
With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme–ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K I) for ATTM as 1.0 ± 0.2 μM and 10.65 μM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.
Keywords: Tyrosinase; Inhibition; Copper chelating; Ammonium tetrathiomolybdate; Whitening agent;