RESUMO
As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 degrees C, in the range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.
Assuntos
Echinococcus granulosus/enzimologia , Lectinas/química , N-Acetilgalactosaminiltransferases/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Células COS/química , Células COS/metabolismo , Bovinos , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/parasitologia , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cobre/fisiologia , DNA Complementar/genética , DNA de Helmintos/genética , Equinococose/enzimologia , Equinococose/veterinária , Proteínas de Helminto/biossíntese , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Concentração de Íons de Hidrogênio , Lectinas/genética , Manganês/metabolismo , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/biossíntese , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/fisiologia , Peptídeos/genética , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Especificidade por Substrato/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (approximately 98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (approximately 76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.
Assuntos
Inibidores de Adenilil Ciclases , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Animais , Células COS/metabolismo , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Isoenzimas/metabolismo , Rim/citologia , Plasmídeos , TransfecçãoRESUMO
Glucocorticoids inhibit insulin expression in cultured pancreatic islet cells. In this study, we provide evidence that transcriptional downregulation of insulin gene expression by glucocorticoids is the result of synergistic interaction between various elements of the insulin promoter. Similar synergistic effects on insulin gene transcription were previously reported for other key insulin regulators, cyclic adenosine monophosphate (cAMP) and glucose. Transfection of CAT constructs containing different segments of the insulin promoter into the pancreatic cell line, HIT T-15 2.2.2, demonstrated that dexamethasone decreased CAT activity in all constructs tested. However, differences were found in the relative sensitivities of the various constructs. Glucocorticoid inhibition of expression from plasmids containing A elements may result from decreased expression of the pancreatic homeodomain protein STF-1. However, a different mechanism must be invoked for insulin promoter constructs lacking A sites, which nevertheless still demonstrated negative regulation. Glucocorticoid-induced inhibition of one of these regions (-882 to -342) was seen to require pancreas-specific factors, because inhibition was observed in HIT T-15 2.2.2 cells but not in the nonpancreatic COS-1 cells. We conclude, therefore, that the human insulin gene contains multiple transcriptional elements that respond to glucocorticoids, some of which require beta cell-specific factors.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Insulina/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Células COS/metabolismo , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , AMP Cíclico/farmacologia , Dexametasona/farmacologia , Sinergismo Farmacológico , Glucose/farmacologia , Humanos , Ilhotas Pancreáticas/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , TransfecçãoAssuntos
Mutagênese Sítio-Dirigida , Plasmídeos/genética , Animais , Células COS/citologia , Células COS/metabolismo , Complemento C4/genética , Enzimas de Restrição do DNA , Vetores Genéticos/genética , Humanos , Ácidos Nucleicos Heteroduplexes/genética , Reação em Cadeia da Polimerase , Polinucleotídeo 5'-Hidroxiquinase , Proteínas Recombinantes de Fusão/genéticaRESUMO
A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.