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1.
Int J Parasitol ; 35(10): 1115-25, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16115636

RESUMO

Cystatins are cysteine protease inhibitors that are widespread in the plant and animal kingdoms. Cystatins are expressed by helminth parasites that may employ these proteins to regulate parasite cysteine protease activity and to modulate host immune responses. Here, we describe the cloning of a cDNA encoding a high molecular weight protein of Fasciola hepatica that contains two domains with significant identity to the cardinal cystatin signatures and four domains with degenerated cystatin signatures. This is the first report of a multi-domain cystatin in an invertebrate species. While cystatins are divided into three evolutionary related families, our phylogenetic analysis shows that all cystatin domains within this protein, like several other helminth cystatins, belong to the cystatin family 2. The DNA region encoding the domain 4 that is the best conserved at the level of its cystatin signatures was expressed in Drosophila cells and a recombinant protein was produced and purified. This protein was a potent inhibitor of the papain and of the major cysteine protease of F. hepatica, the cathepsin L1.


Assuntos
Cistatinas/genética , Fasciola hepatica/genética , Sequência de Aminoácidos , Animais , Southern Blotting/métodos , Catepsinas/antagonistas & inibidores , Clonagem Molecular/métodos , Cistatinas/isolamento & purificação , Inibidores de Cisteína Proteinase/genética , DNA Complementar/genética , Expressão Gênica/genética , Genes de Helmintos/genética , Proteínas de Helminto/genética , Dados de Sequência Molecular , Papaína/antagonistas & inibidores , Filogenia , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência/métodos
2.
Int J Parasitol ; 33(11): 1173-81, 2003 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-13678633

RESUMO

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.


Assuntos
Antígenos de Helmintos/imunologia , Catepsinas/imunologia , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Intestinos/imunologia , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Catepsina L , Catepsinas/genética , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/prevenção & controle , Cisteína Endopeptidases , Fasciolíase/imunologia , Expressão Gênica , Interações Hospedeiro-Parasita , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/prevenção & controle , Vacinas/administração & dosagem
3.
Exp Parasitol ; 104(1-2): 33-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12932757

RESUMO

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.


Assuntos
Dípteros/enzimologia , Proteínas Recombinantes/biossíntese , Serina Endopeptidases/biossíntese , Animais , Bovinos , Células Cultivadas , Cromatografia por Troca Iônica , Drosophila melanogaster/citologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Glicosilação , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Transfecção
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