RESUMO
Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.
Assuntos
Moléculas de Adesão Celular/farmacologia , Galactosídeos/metabolismo , Proteínas de Protozoários/farmacologia , Receptores Toll-Like/agonistas , Toxoplasma/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Galactosídeos/química , Glicoproteínas/química , Células HEK293 , Humanos , Imunidade Inata , Engenharia de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Toxoplasma/genéticaRESUMO
Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides (GOS) from lactose was conducted using a combi-biocatalyst formed by crosslinked enzyme aggregates of Aspergillus oryzae ß-galactosidase and Saccharomyces cerevisiae cells co-immobilized by entrapment in calcium alginate gel particles. Product yield obtained with the combi-biocatalyst was similar than obtained with the soluble enzyme (23.3%), having a final purity of 25.7%. During the simultaneous process, ethyl-ß-galactoside was produced from the ethanol generated as a metabolic product of yeast cells, but ethyl-ß-galactoside was considerably decreased at high aeration (4 vvm). The combi-biocatalyst can be recovered and reused but its performance is limited by the reduction of the metabolic capacity of the cells. In this way, a process was developed for the SSP of GOS from lactose, obtaining a comparable product yield and higher specific productivity than in a conventional synthesis.
Assuntos
Aspergillus oryzae/enzimologia , Enzimas Imobilizadas/metabolismo , Galactose/química , Galactosídeos/metabolismo , Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , beta-Galactosidase/metabolismo , Galactosídeos/isolamento & purificação , Concentração de Íons de Hidrogênio , Oligossacarídeos/isolamento & purificaçãoRESUMO
A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.
Assuntos
Aglutininas/química , Aglutininas/metabolismo , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Holothuria/química , Lectinas/química , Lectinas/metabolismo , Aglutininas/isolamento & purificação , Aglutininas/toxicidade , Sequência de Aminoácidos , Animais , Hemaglutinação , Testes de Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Íons , Lectinas/isolamento & purificação , Lectinas/toxicidade , Lectinas Tipo C , Dados de Sequência Molecular , Coelhos , Alinhamento de Sequência , TemperaturaRESUMO
Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl ß-D-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, K(ic)=0.31±0.04 M and K(iu)=0.604±0.035 M.
Assuntos
Aspergillus oryzae/enzimologia , Galactose/biossíntese , beta-Galactosidase/metabolismo , Catálise , Galactosídeos/metabolismo , Glicosídeo Hidrolases/metabolismoRESUMO
Galectins (GALs) are evolutionarily-conserved lectins defined by at least one carbohydrate recognition domain (CRD) with affinity for beta-galactosides and conserved sequence motifs. Although the biological roles of some members of this family, including the 'proto-type' GAL-1 and the 'chimera-type' GAL-3 have been widely studied, the functions of 'tandem-repeat' galectins are just emerging. The subgroup of 'tandem-repeat' galectins (GAL-4, -6, -8, -9, and -12) contain two distinct CRDs, connected by a linker peptide. Here we integrated and distilled the available information on 'tandem-repeat' galectins, their specific structures, potential ligands and biological activities in inflammatory and neoplastic diseases. While GAL-4 has been implicated in inflammatory bowel diseases, either as a pro-inflammatory or pro-apoptotic mediator, GAL-8 plays roles in autoimmune diseases such as rheumatoid arthritis and lupus erythematosus and modulates tumor progression. GAL-9 controls allergic inflammation and Th1/Th17-mediated autoimmunity and has prognostic value in certain tumor types. Finally, GAL-12 plays important roles in adipocyte physiology. Although this information is just emerging, further studies are needed to dissect the biological roles of 'tandem-repeat' galectins in health and disease.
Assuntos
Galectinas/genética , Sequências de Repetição em Tandem , Animais , Sequência Conservada , Galactosídeos/metabolismo , Galectinas/química , Galectinas/metabolismo , Humanos , Ligantes , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Galectins can recognize and specifically bind to ß-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.
Assuntos
Galectinas/genética , Pinctada/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Etiquetas de Sequências Expressas , Galactosídeos/metabolismo , Galectinas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Imunidade Inata , Dados de Sequência Molecular , Filogenia , Pinctada/classificação , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNA , Sequências de Repetição em TandemRESUMO
The stability of proteins involves a critical balance of interactions of different orders of magnitude. In this work, we present experimental evidence of an increased thermal stability of galectin-1, a multifunctional beta-galactoside-binding protein, upon binding to the disaccharide lactose. Analysis of structural changes occurring upon binding of lectin to its specific glycans and thermal denaturation of the protein and the complex were analyzed by circular dichroism. On the other hand, we studied dimerization as another factor that may induce structural and thermal stability changes. The results were then complemented with molecular dynamics simulations followed by a detailed computation of thermodynamic properties, including the internal energy, solvation free energy, and conformational entropy. In addition, an energetic profile of the binding and dimerization processes is also presented. Whereas binding and cross-linking of lactose do not alter galectin-1 structure, this interaction leads to substantial changes in the flexibility and internal energy of the protein which confers increased thermal stability to this endogenous lectin. Given that an improved understanding of the physicochemical properties of galectin-glycan lattices may contribute to the dissection of their biological functions and prediction of their therapeutic applications, our study suggests that galectin binding to specific disaccharide ligands may increase the thermal stability of this glycan-binding protein, an effect that could influence its critical biological functions.
Assuntos
Galactosídeos/química , Galactosídeos/metabolismo , Galectina 1/química , Sítios de Ligação , Dimerização , Galectina 1/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dobramento de Proteína , TermodinâmicaRESUMO
Derivatives of 5-deoxy-beta-D-galactofuranose (5-deoxy-alpha-L-arabino-hexofuranose) have been synthesized starting from D-galacturonic acid. The synthesis of methyl 5-deoxy-alpha-L-arabino-hexofuranoside (14alpha) was achieved by an efficient strategy previously optimized, involving a photoinduced electron transfer (PET) deoxygenation. Compound 14alpha was converted into per-O-acetyl-5-deoxy-alpha,beta-L-arabino-hexofuranoside (16), an activated precursor for glycosylation reactions. The SnCl4-promoted glycosylation of 16 led to 4-nitrophenyl (19alpha), and 4-methylthiophenyl 5-deoxy-alpha-L-arabino-hexofuranosides (20alpha). The oxygenated analog 4-methylphenyl 1-thio-beta-D-galactofuranoside (23beta) was also prepared. The 5-deoxy galactofuranosides were evaluated as inhibitors or substrates of the exo-beta-D-galactofuranosidase from Penicillium fellutanum, showing that the absence of HO-5 drastically diminishes the affinity for the protein.
Assuntos
Galactosídeos/química , Galactosídeos/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Penicillium/enzimologia , Galactosídeos/síntese química , Especificidade por SubstratoRESUMO
We developed a new method for the quantification of parasites in tissue. Trypanosoma cruzi strain CL parasites were genetically engineered to express the Escherichia coli beta-galactosidase gene, lacZ and this enzyme is able to catalyze a colorimetric reaction with chlorophenol red beta-D: galactopyranoside (CPRG) as the substrate. The animals were infected with clone CL Brener strain B5 of T. cruzi and treated with benznidazole in order to verify the reduction in the number of parasites in tissue study by quantifying the enzyme beta-galactosidase. The assay demonstrates a reduction in the number of parasites in the groups treated. Thus, this test can be used to test other substances with the aim of verifying the effectiveness in the chronic phase of experimental Chagas' disease.
Assuntos
Doença de Chagas/parasitologia , Trypanosoma cruzi/isolamento & purificação , Animais , Clorofenóis/metabolismo , Colorimetria/métodos , Escherichia coli/enzimologia , Galactosídeos/metabolismo , Genes Reporter/genética , Camundongos , Camundongos Endogâmicos BALB C , Coloração e Rotulagem/métodos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.