RESUMO
Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.
Assuntos
Antivirais , Clorófitas , Lectinas , Polissacarídeos , Animais , Humanos , Antivirais/farmacologia , Antivirais/química , Clorófitas/química , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Lectinas/farmacologia , Lectinas/química , Lectinas/metabolismo , Lectinas/isolamento & purificação , Polissacarídeos/farmacologia , Polissacarídeos/químicaRESUMO
Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.
RESUMO
Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.
RESUMO
We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.
Assuntos
COVID-19 , Orthomyxoviridae , Humanos , SARS-CoV-2/metabolismo , Manose/metabolismo , Fucose , Lectinas/farmacologia , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Polissacarídeos/metabolismoRESUMO
The fate of strontium-90 (90Sr) from water to aquatic biota is of concern since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident because of continuous small 90Sr releases to the seawater from the FDNPP site. The Japanese diet includes many edible marine and freshwater species, and the environmental parameter, that is, the concentration ratio (CR) from water to biota, is useful to estimate the potential 90Sr intake, especially from frequently consumed seafoods. However, widely used CR data for radiation dose assessment only have provided values for biota types such as fish, crustaceans, macroalgae, and so forth, and thus, it is difficult to identify specific data for each species. In this study, therefore, we collated CR data of Sr for aquatic biota by surveying available open data sources from the 1950s to 2019, not only for edible parts but also for whole and inedible parts. In total, we obtained 3800 CR data: 3013 data for marine biota, 28 data for brackish water biota, and 759 data for freshwater biota. The results showed that species-specific CRs may decrease the uncertainties compared to those published in IAEA documents; however, different diets and living areas by species may lead to different uncertainties for different species.
Assuntos
Acidente Nuclear de Fukushima , Monitoramento de Radiação , Poluentes Radioativos da Água , Animais , Biota , Radioisótopos de Césio/análise , Água Doce , Japão , Estrôncio , Água , Poluentes Radioativos da Água/análiseRESUMO
We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10-11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family.
Assuntos
Antivirais/isolamento & purificação , Clorófitas/química , Hemaglutininas Virais/metabolismo , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Lectinas/farmacologia , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/farmacologia , Manose/química , Sequência de Aminoácidos , Antivirais/farmacologia , Monossacarídeos/farmacologia , Oligossacarídeos/química , Polissacarídeos/farmacologia , Ligação Proteica , Rodófitas/química , Internalização do Vírus/efeitos dos fármacosRESUMO
Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Lectinas de Ligação a Manose/farmacologia , Rodófitas/química , Animais , Antivirais/química , Antivirais/isolamento & purificação , Cães , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Lectinas/química , Lectinas/isolamento & purificação , Lectinas/farmacologia , Células Madin Darby de Rim Canino , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/isolamento & purificação , Internalização do Vírus/efeitos dos fármacosRESUMO
Tetrodotoxin (TTX), or pufferfish toxin, has been frequently detected in edible bivalves around the world during the last decade and is problematic in food hygiene and safety. It was reported recently that highly concentrated TTX was detected in the midgut gland of the akazara scallop Chlamys (Azumapecten) farreri subsp. akazara collected in coastal areas of the northern Japanese archipelago. The toxification of the bivalve was likely to involve the larvae of the flatworm, Planocera multitentaculata. However, the overall status of bivalve TTX toxification has not been elucidated. In this study, 14 species/subspecies of bivalves from various Japanese waters were subjected to LC-MS/MS analysis to reveal TTX toxification state, demonstrating that the Pectinidae, including C. farreri akazara, Chlamys farreri nipponensis, Chlamys (Mimachlamys) nobilis, and Mizuhopecten yessoensis, accumulated TTX in their midgut gland. Many individuals of C. farreri akazara and C. farreri nipponensis were found with high concentrations of TTX, while C. nobilis and M. yessoensis exhibited low concentrations. The extent of TTX accumulation in C. farreri akazara and C. farreri nipponensis varied widely by region and season. Curiously, no other bivalve species investigated in this study showed evidence of TTX. These results suggest that monitoring for TTX, like other shellfish toxins, is necessary to ensure that pectinid bivalves are a safe food resource.
Assuntos
Pectinidae , Platelmintos , Tetrodotoxina , Animais , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Tetrodotoxina/análiseRESUMO
The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1-2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1-2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1-2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 10(8) M(-1)) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent.
Assuntos
Clorófitas/química , HIV-1/fisiologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/farmacologia , Manose/química , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Clorófitas/genética , Clorófitas/metabolismo , Cães , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Manose/genética , Manose/metabolismo , Lectinas de Ligação a Manose/genética , Dados de Sequência Molecular , Proteínas de Plantas/genéticaRESUMO
A novel lectin (CBA) was isolated from the green alga, Codium barbatum, by conventional chromatographic methods. The hemagglutination-inhibition profile with sugars and glycoproteins indicated that CBA had preferential affinity for complex type N-glycans but not for monosaccharides, unlike the other known Codium lectins specific for N-acetylgalactosamine. CBA consisted of an SS-linked homodimer of a 9257-Da polypeptide containing seven cysteine residues, all of which were involved in disulfide linkages. The cDNA of the CBA subunit coded a polypeptide (105 amino acids) including the signal peptide of 17 residues. The calculated molecular mass from the deduced sequence was 9705 Da, implying that the four C-terminal amino acids of the CBA proprotein subunit were post-translationally truncated to afford the mature subunit (84 amino acids). No significantly similar sequences were found during an in silico search, indicating CBA to be a novel protein. CBA is the first Codium lectin whose primary structure has been elucidated.
Assuntos
Proteínas de Algas/genética , Clorófitas/química , Lectinas/genética , Subunidades Proteicas/genética , Proteínas de Algas/isolamento & purificação , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Dimerização , Dissulfetos/química , Escherichia coli , Testes de Inibição da Hemaglutinação , Lectinas/isolamento & purificação , Lectinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.
Assuntos
Poríferos , Receptores de Trombopoetina , Animais , Lectinas , Poríferos/metabolismo , Receptores de Trombopoetina/metabolismo , Açúcares , TrombopoetinaRESUMO
The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration-HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1-3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC50s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/virologia , Lectinas/farmacologia , Lectinas de Ligação a Manose/farmacologia , Rodófitas , Animais , Antivirais/química , Linhagem Celular , Embrião de Galinha , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Lectinas/química , Lectinas/metabolismo , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismoRESUMO
High mannose (HM)-binding Oscillatoria agardhii agglutinin homologue (OAAH) lectin family is an important class of anti-viral proteins. The OAAH family lectins show potent anti-influenza virus activity with EC50 of nanomolar levels by binding to HM glycans of the envelope glycoprotein hemagglutinin (HA), thereby inhibiting the viral entry into host cells. No broadly effective neutralizing vaccines for influenza virus are available due to the frequent antigenic drift caused by rapid mutations. Alternatives for vaccines need to be developed to prepare for a possible risk of future emergence of a highly virulent virus. Possible use of antiviral lectins is a simple and useful strategy to prevent viral infection by interfering with the interaction between viral HA and the host sialic acid-containing receptor. High-density glycans of surface HA are primary targets for the lectins to inhibit viral entry. In general, the anti-influenza virus potency of lectins is evaluated by a series of inhibitory assays for infection, such as neutral red dye uptake assay to determine the extent of viral cytopathic effect, and immunofluorescence microscopy to detect the expression of viral proteins in infected cells. Direct interaction between lectins and HA could be evaluated by enzyme-linked immunosorbent assay or surface plasmon resonance analysis.
Assuntos
Antivirais/farmacologia , Proteínas de Bactérias/farmacologia , Lectinas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cães , Humanos , Lectinas/química , Lectinas/metabolismo , Células Madin Darby de Rim Canino , Manose/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Orthomyxoviridae/fisiologia , Planktothrix/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
A microarray comprising 3,514 cDNAs was constructed from a medaka EST library to elucidate the transcriptional responses associated with temperature shift from 25 to 15 degrees C in a medaka cell line. Microarray analysis revealed that the mRNA levels of 313 clones were significantly different in at least one combination of different incubation periods up to 7 days at a given incubation temperature or between 25 and 15 degrees C at a given incubation period (P < 0.05). These genes are known to be associated with various biological processes including morphogenesis, cell proliferation and response to stress. A number of genes encoding proteins which localize in extracellular areas were apparently up-regulated at 15 degrees C, whereas those localizing in intracellular areas were down-regulated at this temperature. In addition, while a number of genes represented long-term expression changes, only a few responded to short-term inductions. A typical example was CYR61, a multifunctional matricellular signaling modulator, the mRNA levels of which increased after temperature shift from 25 to 15 degrees C in 3 h, and then decreased rapidly to near the original level within 12 h. Another series of analyses by quantitative reverse transcription-PCR revealed that the mRNA levels of CYR61 at 5 degrees C were significantly higher even at 24 h after temperature shift compared to those of the cells successively maintained at 25 degrees C. These analyses suggest that remodeling and reorganizing of extracellular structure of cells are important to offset the low temperature effect and CYR61 is considered to be a novel gene associated with temperature response in poikilotherms.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Peixes/genética , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Oryzias/genética , Temperatura , Animais , Linhagem Celular , Proteínas de Peixes/fisiologia , Perfilação da Expressão Gênica , Genômica , Proteínas Imediatamente Precoces/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Metabolismo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estações do AnoRESUMO
In brown macroalgae, alginate and D-mannitol are promising carbohydrates for biorefinery. Saccharomyces cerevisiae is widely used as a microbial cell factory, but this budding yeast is unable to utilize either alginate or D-mannitol. Alginate can be depolymerized by both endo-type and exo-type alginate lyases, yielding a monouronate, 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a key intermediate in the metabolism of alginate. Here, we constructed engineered two S. cerevisiae strains that are able to utilize both DEH and D-mannitol on two different strain backgrounds, and we also improved their aerobic growth in a DEH liquid medium through adaptive evolution. In both evolved strains, one of the causal mutations was surprisingly identical, a c.50A > G mutation in the codon-optimized NAD(P)H-dependent DEH reductase gene, one of the 4 genes introduced to confer the capacity to utilize DEH. This mutation resulted in an E17G substitution at a loop structure near the coenzyme-binding site of this reductase, and enhanced the reductase activity and aerobic growth in both evolved strains. Thus, the crucial role for this reductase reaction in the metabolism of DEH in the engineered S. cerevisiae is demonstrated, and this finding provides significant information for synthetic construction of a S. cerevisiae strain as a platform for alginate utilization.
Assuntos
Alginatos/metabolismo , Evolução Molecular Direcionada , Engenharia Genética , Hexoses/metabolismo , Oxirredutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Carbono/farmacologia , Etanol/metabolismo , Fermentação , Cinética , Manitol/metabolismo , Mutação/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Two types of the fast skeletal myosin heavy chain (MYH) genes were cloned from a genomic DNA library of carp (Cyprinus carpio L.) and named MYH10 and MYH30, which showed the sequence similarity to the MYH cDNAs predominantly expressed in carp acclimated to 10 and 30 degrees C, respectively. The 5'-flanking region of about 3 kbp in size each from MYH10 and MYH30 contained various cis-elements to bind to transcriptional regulatory factors such as MyoD family and myocyte enhancer factor 2 (MEF2) family members. To localize functional regions responsible for the MYH gene expression in a temperature-dependent manner, a series of deletion constructs were prepared from the 5'-flanking region, inserted upstream the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30 degrees C. The sequence of -1004 to -995 bp with the transcriptional activity in MYH30 was identified as an MEF2 binding site. While the activity given by a sequence of -921 to -824 bp in MYH10 contained only a GATA box, that of the activity of the -1 kbp construct from MYH10 was markedly higher in carp reared at 10 degrees C than fish reared at 30 degrees C. On the other hand, no temperature-dependent expressional regulation was observed for MYH30 even with the full-length construct of -3 kbp. The DNA fragment of -921 to -824 bp in MYH10 and MEF2 binding site in MYH30 interacted with nuclear proteins extracted from carp fast skeletal muscle as revealed by electrophoretic mobility shift assay. The signal intensity of a complex formed between the DNA fragment of MYH10 and nuclear extracts from the 10 degrees C-acclimated carp were higher than those with extracts from the 30 degrees C-acclimated fish. Although MEF2-binding site in MYH30 could form complex with nuclear extracts from the 30 degrees C-acclimated carp, the same or stronger signals were detected in complex formed with extracts from the 10 degrees C-acclimated fish.
Assuntos
Região 5'-Flanqueadora/genética , Peixes/genética , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Miosina não Muscular Tipo IIB/genética , Temperatura , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Clonagem Molecular , Genes Reporter , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Miosina não Muscular Tipo IIB/química , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência/genética , Homologia de Sequência de AminoácidosRESUMO
Alginate, a major acidic polysaccharide in brown macroalgae, has attracted attention as a carbon source for production of ethanol and other chemical compounds. Alginate is monomerized by exo-type alginate lyase into an unsaturated uronate; thus, this enzyme is critical for the saccharification and utilization of alginate. Although several exo-type alginate lyases have been characterized independently, their activities were not assayed under the same conditions or using the same unit definition, making it difficult to compare enzymatic properties or to select the most suitable enzyme for saccharification of alginate. In this study, we characterized the three bacterial exo-type alginate lyases under the same conditions: A1-IV of Sphingomonas sp. strain A1, Atu3025 of Agrobacterium tumefaciens, and Alg17c of Saccharophagus degradans. A1-IV had the highest specific activity as well as the highest productivity of uronate, whereas Alg17c had the lowest activity and productivity. Only dialyzed Atu3025 and Alg17c were tolerant to freezing. Alg17c exhibited a remarkable halotolerance, which may be advantageous for monomerization of alginate from marine brown algae. Thus, each enzyme exhibited particular desirable and undesirable properties. Our results should facilitate further utilization of the promising polysaccharide alginate.
Assuntos
Bactérias/enzimologia , Polissacarídeo-Liases/metabolismo , Estabilidade Enzimática , Polissacarídeo-Liases/química , Polissacarídeo-Liases/isolamento & purificaçãoRESUMO
We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).
Assuntos
Proteínas de Algas/farmacologia , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Lectinas/farmacologia , Rodófitas/química , Internalização do Vírus/efeitos dos fármacos , Proteínas de Algas/biossíntese , Proteínas de Algas/genética , Proteínas de Algas/isolamento & purificação , Sequência de Aminoácidos , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/metabolismo , Sequência de Carboidratos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Humanos , Células Jurkat , Lectinas/biossíntese , Lectinas/genética , Lectinas/isolamento & purificação , Manose/química , Manose/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV/fisiologia , Lectinas de Ligação a Manose/administração & dosagem , Lectinas de Ligação a Manose/metabolismo , Rodófitas/metabolismo , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sítios de Ligação , HIV/efeitos dos fármacos , Lectinas de Ligação a Manose/genética , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Rodófitas/genéticaRESUMO
Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.