RESUMO
Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N. americanus cDNA library for the presence of hookworm allergens. Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli. Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host. Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.
Assuntos
Alérgenos/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Necator americanus/imunologia , Ribonucleoproteínas/imunologia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Calreticulina , Clonagem Molecular , Reações Cruzadas/imunologia , Escherichia coli/genética , Biblioteca Gênica , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Necator americanus/genética , Necator americanus/crescimento & desenvolvimento , Necatoríase/sangue , Necatoríase/imunologia , Necatoríase/parasitologia , Papua Nova Guiné , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Procathepsin B from the parasitic trematode Schistosoma mansoni was expressed as a glycosylation-minus mutant in yeast cells and purified by means of a histidine affinity tag which was added to the carboxyl terminus of the recombinant protein. The purified zymogen underwent autoprocessing but required an assisting protease for activation. Pepsin-activated schistosomal cathepsin B was further characterized with the cathepsin B-specific substrates N-benzyloxycarbonyl (Z)-Arg-Arg-p-nitroanilide, Z-Arg-Arg-7-amido-4-methyl-coumarin, and Z-Phe-Arg-7-amido-4-methylcoumarin. A proteolytic activity comparable to mammalian cathepsin B was observed. In addition, we analyzed the degradation of human hemoglobin by schistosomal cathepsin B, which has been suggested to be the physiological target of the protease.
Assuntos
Catepsina B/isolamento & purificação , Catepsina B/metabolismo , Precursores Enzimáticos/metabolismo , Hemoglobinas/metabolismo , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina B/biossíntese , Clonagem Molecular , Primers do DNA , Ativação Enzimática , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/isolamento & purificação , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pepsina A/metabolismo , Plasmídeos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The effect of deletion of each of the two authentic polyprotein translation initiation sites of foot-and-mouth disease virus on viral protein synthesis and replication was analyzed. Deletion of either the first or the second initiation site led to the expression of only one form of the leader protein, L or L', respectively, but in vitro processing of the viral polyprotein and cleavage of eIF-4 gamma were not affected by either deletion. Whereas RNA in which the first translation initiation site had been deleted led to the production of viruses in transfected BHK cells, deletion of the second translation initiation site abolished virus replication.
