Expression of Cryptosporidium parvum Cpa135/CpCCP1 chimeras in Giardia duodenalis: organization of the protein domains affects the protein secretion pathway.
Exp Parasitol
; 127(3): 680-6, 2011 Mar.
Article
em En
| MEDLINE
| ID: mdl-21112325
ABSTRACT
Cpa135 is a multidomain antigenic protein secreted at the sporozoite stage of the Apicomplexa protozoan Cryptosporidium parvum. Previous studies have shown that the protozoan flagellate parasite Giardia duodenalis is a suitable system for the heterologous expression of secreted proteins of Apicomplexa. Here, we designed three different Cpa135 variants fused to a C-terminal HA tag in order to test their expression in G. duodenalis under the control of the inducible promoter of the cyst wall protein 1 gene (cwp1). The three Cpa135 chimeras encompassed different portions of the protein; CpaG encodes the entire polypeptide of 1574 amino acids (aa); CpaGΔC includes the first 826 aa at the N-terminus; and CpaGΔN consists in of the final 833 aa at the C-terminus. Immunoblot experiments showed that CpaG and CpaGΔN maintained the epitopes recognized by anti-C. parvum-specific human serum. The intracellular localization and transport of the three Cpa135 variants were studied by immunofluorescence in combination with G. duodenalis-specific antibodies. CpaGΔC was mainly accumulated in the endoplasmic reticulum and the intact form was also excreted in the medium. Differently, the Cpa135 chimeras possessing an intact C-terminus (CpaG and CpaGΔN) were transported towards the forming cyst wall possibly and were not detected in the medium. Furthermore, the full-length CpaG was incorporated into the cyst wall. The data presented suggest that the C-terminus of Cpa135, which includes a cysteine reach domain, could influence the secretion of the chimeric proteins.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
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Glicoproteínas de Membrana
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Cryptosporidium parvum
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Giardia
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article