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Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

ABSTRACT: The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

SUBMITTER: Gilk SD

PROVIDER: S-EPMC1595352 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

Gilk Stacey D SD Raviv Yossef Y Hu Ke K Murray John M JM Beckers Con J M CJ Ward Gary E GE

Eukaryotic cell 20061001 10

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylpho ...[more]

PMID: 17030994

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Project description:Toxoplasma gondii is an obligate intracellular parasite. The ability to invade cells and proliferate successfully is given by the machinery found in the pellicle of the parasite, the pellicle is a complex of three membranes. Despite the importance of the pellicle in the biology of Toxoplasma, its protein composition had not been reported. The proteins of the isolated pellicle fraction were digested with trypsin and chymotrypsin, for their identification and quantification in the case of those digested with trypsin and only identification in the case of those digested with chymotrypsin. Samples were resolved, and peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry using the high-definition multiplexed MS/MS mode. We used the Hi3 method to quantify absolutely every protein detected. In the isolate of pellicle digested with trypsin a total of 548 proteins were identified and absolutely quantified using the internal standard and the intensity of the most abundant peptides per protein with Progenesis QI software and the database UP000002494. Among identified proteins, 11 corresponded to IMC, 11 were related to SAG (SRS), 15 to plasma membrane and glideosome and 13 to cytoskeleton (CK). In the chymotrypsin-digested pellicle sample a total of 137 proteins were identified and 22 of them were proteins that were not identified in the trypsin-digested sample. Due to the characteristics of the SRS proteins, they have a role in generating the host's immune response or are involved in adhesion, motility and invasion. The 11 proteins found with the analysis of E. mass. Study of these SRS proteins to determine their role in the dynamic events of the parasite, such as adhesion and motility as well as in the generation of the immune response, is important in the search for therapies against toxoplasmosis

Dataset Information

Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

Publications

Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide.

Similar Datasets

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